ES2761448T3 - Procedure for enzymatic hydrolysis of lignocellulosic material with oxygen addition - Google Patents

Abstract

Procedure for the preparation of a sugar product from lignocellulosic material, comprising the following steps: a) optionally, pretreatment of the lignocellulosic material, b) optionally, washing of the optionally pretreated lignocellulosic material, c) enzymatic hydrolysis of the optionally washed lignocellulosic material and / or optionally pretreated using an enzyme composition comprising at least two cellulases and whereby the enzyme composition comprises at least GH61, and d) optionally, recovery of the sugar product, wherein, for part of the hydrolysis time enzymatic, oxygen is added to the lignocellulosic material, and for part of the time of enzymatic hydrolysis less or no oxygen is added to the lignocellulosic material compared to the other part of the time of enzymatic hydrolysis, the reactor for enzymatic hydrolysis has a volume 1 m3 or more, and the dry matter content in the hydrolysis stage (c) it is 10% by weight or more, in which the part of the time in which more oxygen is added is 12 to 50% when the oxygen is added in the second half time of the enzymatic hydrolysis, or 2 to 30 % of the total time of enzymatic hydrolysis when oxygen is added in the first half of the time of enzymatic hydrolysis, and thus the concentration of oxygen in the liquid phase of hydrolysis during the part of the time in which oxygen is added it is at least 2 times the concentration of oxygen in the liquid phase during the part of the time in which less or no oxygen is added.

Description

DESCRIPTION

Procedure for enzymatic hydrolysis of lignocellulosic material with oxygen addition

Field of the Invention

The present invention relates to a process for the enzymatic hydrolysis of lignocellulosic material and fermentation of sugars, in which oxygen is added during part of the time of the enzymatic hydrolysis.

Background of the Invention

Lignocellulosic plant material, also referred to here as raw material, is a renewable energy source in the form of sugars that can be converted into valuable products, for example sugars or biofuel, such as bioethanol. During this procedure, the (ligno or hemi) cellulose present in the raw material, such as wheat straw, corn stubble, rice husks, etc., is converted to reducing sugars by (hemi) cellulolytic enzymes, which are then converted optionally in valuable products such as ethanol by microorganisms such as yeasts, bacteria and fungi.

Since (hemi) -cellulose is crystalline and trapped in a lignin network, conversion to reducing sugars is generally slow and incomplete. Typically, enzymatic hydrolysis of the untreated feedstock produces sugars in an amount <20% of theory. Applying a chemical and thermo-physical pretreatment, (hemi) cellulose is more accessible for (hemi) -cellulolytic enzymes, and thus, conversions are faster and with higher yields.

A typical yield of ethanol from glucose, derived from pretreated corn stubble, is 151.4 liters (40 gallons) of ethanol per 1000 kg of dry corn stubble (Badger, P, Ethanol from cellulose: a general review, Trends in new crops and new uses, 2002, J. Janick and A. Whipkey (eds.) ASHS Press, Alexandria, VA) or 0.3 g of ethanol per g of raw material. The maximum yield of ethanol in a cellulose base is approximately 90%.

Cellulolytic enzymes - produced for the most part by species such as Trichoderma, Humicola and Aspergillus - are used commercially to convert the pretreated raw material into a mash containing insoluble (hemi) cellulose, reducing sugars obtained therefrom, and lignin. Rasamsonia- derived thermostable cellulolytic enzymes have been used to degrade lignocellulosic feedstock, and these enzymes are known for their thermostability, see WO2007091231. The mash produced is used in a fermentation during which reducing sugars are converted into yeast biomass (cells), carbon dioxide and ethanol. The ethanol produced in this way is called bioethanol.

The usual production of sugars from pretreated lignocellulosic feedstock, hydrolysis also called liquefaction, pre-saccharification or saccharification, typically takes place during a procedure lasting 6 to 168 hours (Kumar, S., Chem. Eng. Technol. 32 (2009) 517-526) at elevated temperatures of 45 to 50 ° C and in non-sterile conditions. During this hydrolysis, the cellulose present is partially converted (typically 30 to 95%, depending on the enzymatic activity and the conditions of hydrolysis) into reducing sugars. In the case of enzyme inhibition by compounds present in the pretreated raw material and by released sugars; and to minimize thermal inactivation, this period of elevated temperature is minimized as much as possible.

Fermentation following hydrolysis takes place in a different process step, preferably anaerobic, either in the same container or in a different container, in which the temperature is adjusted to 30 to 33 ° C (mesophilic procedure) to suit to the growth and production of ethanol by microbial biomass, commonly yeasts. During this fermentation procedure, the remaining (hemi) cellulosic material is converted to reducing sugars by the enzymes already present from the hydrolysis stage, while producing microbial biomass and ethanol. Fermentation is complete once the (hemi) cellulosic material is converted to fermentable sugars, and all fermentable sugars are converted to ethanol, carbon dioxide, and microbial cells. This can take up to 6 days. In general, the overall process time for hydrolysis and fermentation can be up to 13 days.

The fermented mash obtained in this way consists of non-fermentable sugars, non-hydrolyzable (hemi) cellulosic material, lignin, microbial cells (most commonly yeast cells), water, ethanol, dissolved carbon dioxide. During the successive steps, the ethanol is distilled from the mash and further purified. The remaining solid suspension is dried and used, for example, as combustion fuel, fertilizer, or livestock feed.

W02010080407 suggests treating cellulosic material with a cellulase composition under anaerobic conditions. The removal or exclusion of reactive oxygen species can improve the performance of cellulose hydrolyzing enzyme systems. The hydrolysis of cellulosic material, for example lignocellulose, by an enzyme composition can be reduced by oxidative damage to the components of the enzyme composition and / or oxidation of the cellulosic material, for example by molecular oxygen.

WO2009046538 describes a method of treating plant materials as a lignocellulosic raw material for releasing fermentable sugars using an enzymatic hydrolysis procedure to treat the materials under vacuum and producing a sugar rich process stream comprising reduced amounts of volatile sugar / inhibitor compounds from fermentation, such as furfural and acetic acid. Aside from removing volatile inhibitor compounds, other compounds and / or molecules that are also removed include nitrogen, oxygen, argon, and carbon dioxide.

Podkaminer et al., Biotechnology for Biofuels 2012, 5:43, describe a previously unrecognized incompatibility of enzymes secreted by an aerobic fungus with the fermentation conditions of an anaerobic bacterium, and suggest that they would be valuable enzymes more suitable for industrially fermentation conditions relevant.

Canella et al., Biotechnology for Biofuels 2012, 5:26, describe that the presence of oxidative enzymes in Cellic Ctec-2 led to the formation of cellobionic and gluconic acid during the hydrolysis of pretreated wheat straw and filter paper.

Canella et al., Biotechnology & Bioengineering 2013, 111: 59, describe the impact of a selected enzyme preparation and the processing strategy on the final yield of ethanol and the global behavior with pretreated wheat straw up to 30% Dm.

Kumar et al., Final Technical Report 2012 (retrievable at https://www.osti.gov/servlets/purl/1068167), describe the development of a commercial enzyme system for saccharification of lignocellulosic biomass.

With each batch of feedstock, enzymes are added to maximize the yield and rate of fermentable sugars released from the pretreated lignocellulosic feedstock during the given procedure time. In general, costs for enzyme production, ethanol raw material yields, and investments are the main cost factors in overall production costs (Kumar, S. Chem. Eng. Technol. 32 (2009) 517- 526). Until now, the cost for reducing the use of enzymes has been achieved by applying enzyme products from a single or multiple microbial sources (document WO 2008/008793) with broader and / or greater (specific) hydrolytic activity, the use of which is intended to reduce the need of enzyme, faster conversion rates and / or higher conversion yields, and thus lower overall bioethanol production costs. This requires large investments in research and development of these enzyme products. In the case of an enzyme product composed of enzymes from multiple microbial sources, large capital investments are required for the production of each individual enzyme compound.

Therefore, it is desirable to improve the above procedure involving hydrolysis and fermentation.

Summary of the invention

Therefore, an object of the invention is to provide a process in which the hydrolysis step is carried out under improved conditions. Another object of the invention is to provide a process involving hydrolysis that has a reduced process time. A further object of the invention is to provide a process in which the enzyme dosage can be reduced and, at the same time, the production of a useful hydrolysis product is kept at the same level, or even increased. Another object is to provide a process involving hydrolysis, in which the conditions of the hydrolysis process are optimized. A still further object of the invention is to provide a process involving hydrolysis, in which the production of a useful hydrolysis product is increased using the same dosage of enzyme. One or more of these objects are achieved in accordance with the invention.

The present invention provides a process for the preparation of a sugar product from lignocellulosic material, comprising the following steps:

a) optionally, pretreating the lignocellulosic material;

b) optionally washing the optionally pretreated lignocellulosic material;

c) enzymatically hydrolyzing the optionally washed and / or optionally pretreated lignocellulosic material using an enzyme composition comprising at least two cellulases and whereby the enzyme composition comprises at least GH61; and

d) optionally, recovering a sugar product;

wherein for part of the enzymatic hydrolysis time, oxygen is added to the lignocellulosic material, and for part of the enzymatic hydrolysis time less or no oxygen is added to the lignocellulosic material compared to the other time part of the hydrolysis enzymatic, the reactor for enzymatic hydrolysis has a volume of 1 m3 or more, and the dry matter content in the hydrolysis step (c) is 10% by weight or more, in which the part of the time in which adds more oxygen is 12 to 50% when oxygen is added in the second half of the time of enzymatic hydrolysis, or 2 to 30% of the total time of enzymatic hydrolysis when oxygen is added in the first half of the time of enzymatic hydrolysis , and so the concentration of oxygen in the liquid phase of hydrolysis during the part of the time in which oxygen is added is at least 2 times the concentration of oxygen in the liquid phase during the part of time in which it is added less oxygen or no oxygen is added.

Additionally, the present invention provides a process for the preparation of a fermentation product from lignocellulosic material, which comprises the following steps:

a) optionally, pretreating the lignocellulosic material;

b) optionally washing the optionally pretreated lignocellulosic material;

c) enzymatically hydrolyzing the optionally washed and / or optionally pretreated lignocellulosic material using an enzyme composition comprising at least two cellulases and whereby the enzyme composition comprises at least GH61;

d) fermenting the hydrolyzed lignocellulosic material, to produce a fermentation product; and

e) optionally, recovering a fermentation product;

wherein for part of the time of enzymatic hydrolysis, oxygen is added to the lignocellulosic material, and for part of the time of enzymatic hydrolysis less or no oxygen is added to the lignocellulosic material compared to the other part of the time of hydrolysis enzymatic, the reactor for enzymatic hydrolysis has a volume of 1 m3 or more, and the dry matter content in the hydrolysis step (c) is 10% by weight or more, in which the part of the time in which adds more oxygen is 12 to 50% when oxygen is added in the second half of the time of enzymatic hydrolysis, or 2 to 30% of the total time of enzymatic hydrolysis when oxygen is added in the first half of the time of enzymatic hydrolysis , and so the concentration of oxygen in the liquid phase of hydrolysis during the part of the time in which oxygen is added is at least 2 times the concentration of oxygen in the liquid phase during the part of time in which it is added Ade less oxygen or no oxygen is added.

According to a preferred embodiment of the invention, the part of the time in which less or preferably no oxygen is added is 10 to 80%, preferably 20 to 80%, more preferably 30 to 80%, and most preferably 40 80% of the total time of the enzymatic hydrolysis.

According to another preferred embodiment of the invention, the part of the time in which more oxygen is added is preferably 8 to 50%, and most preferably, 10 to 50% of the total time of the enzymatic hydrolysis, more preferably the part of the time when more oxygen is added is

a) preferably 20 to 40% when oxygen is added in the second half time of the enzymatic hydrolysis;

b) preferably 4 to 25%, and more preferably 5 to 20% of the total enzymatic hydrolysis time when oxygen is added in the first half of the enzymatic hydrolysis time.

Advantageously, the concentration of oxygen in the liquid phase of hydrolysis during the part of the time in which oxygen is added is preferably at least 4 times, more preferably at least 10 times the concentration of oxygen in the liquid phase during the part of time in which less or no oxygen is added.

According to a further preferred embodiment of the invention, in the part of the time when oxygen is added, the concentration of oxygen in the liquid phase, in which the lignocellulosic material is present during enzymatic hydrolysis, is at least 0.001 mol / m3, preferably at least 0.002 mol / m3, and most preferably at least 0.003 mol / m3, and even more preferably more than 0.01 mol / m3, for example more than 0.02 mol / m3 or 0.03 mol / m3. In reactors of less than 1 m3, OD values below 0.01 mol / m3 or 0.02 mol / m3 will be obtained with slow stirring. Vigorous mixing or stirring on such a scale introduces part of the gaseous phase of the headspace into the reaction liquid. For example, mixing or stirring can create a swirl that draws oxygen into the liquid. In general, flushing the headspace with oxygen (eg in the form of air) in combination with (vigorous) mixing or stirring will introduce sufficient oxygen into the cellulosic material in the hydrolysis reactor for reactors with a size of 100 liters up to 1 m3 . On a larger scale, for example in a 50 m3 or larger reactor, for example 100 m3, so much energy is needed for vigorous stirring that, economically, this will not apply in a commercially operating process. In general, in large reactors, agitation or mixing without introduction of air or oxygen will result in OD values of less than 0.01 mole / m3.

According to still another preferred embodiment of the invention, during the addition of oxygen (in the part of the time when oxygen is added), the concentration of oxygen in the liquid phase, in which the lignocellulosic material is present during enzymatic hydrolysis , is preferably at most 80% of the oxygen saturation concentration under the conditions of the hydrolysis reaction, more preferably at most 0.12 mol / m3, even more preferably at most 0.09 mol / m3, even more preferably as maximum 0.06 mol / m3, even more preferably still maximum 0.045 mol / m3, and most preferably, maximum 0.03 mol / m3. Temperature and pressure will influence DO. The preferred and exemplary values in mol / m3 given above refer to normal atmospheric pressure and a temperature of approximately 62 ° C. One skilled in the art will appreciate favorable OD values based on the present teachings.

The enzymatic hydrolysis time of the present process is preferably from 5 to 150 hours. According to a further preferred embodiment of the invention, the enzyme composition is derived from a fungus, preferably a microorganism of the Rasamsonia genus , or the enzyme composition comprises a fungal enzyme, preferably a Rasamsonia enzyme . According to still another preferred embodiment of the invention, the dry matter content in the hydrolysis step c) is 14% by weight or more, and even more preferably it is 14 to 33% by weight. The enzymatic hydrolysis preferably takes place in a batch, batch and / or continuous culture reactor. Preferably, the oxygen that is introduced in the present process is an oxygen-containing gas, such as air. By "less oxygen is added to or is present in the lignocellulosic material during part of the time of enzymatic hydrolysis" is meant that at least 50% less, preferably at least 70% less, most preferably at least 90 is introduced or present % less oxygen (expressed in mol of oxygen / m3), for example in the form of bubbles, than that which is added or is present during the other part of the enzymatic hydrolysis time in which less oxygen is added.

In a preferred embodiment, oxygen is added in the form of bubbles (soda).

Surprisingly, according to the invention, by adding oxygen it is possible to achieve many process advantages, including optimal temperature conditions, reduced process time, reduced enzyme dosage, enzyme reuse, higher yields and other process optimizations, which result in reduced costs.

In one embodiment, the stable enzyme composition used retains activity for 30 hours or more. According to a further embodiment, the hydrolysis is preferably carried out at a temperature of 40 ° C or more, more preferably at a temperature of 50 ° C or more, and most preferably, at a temperature of 55 ° C or more. The process of the invention will be illustrated in more detail below.

Brief description of the figures

Fig. 1: The effect of bubbling nitrogen or air through a 10% aCS raw material before hydrolysis, on the total amount of glucose (g / l) released by the TEC-210 mixture (1), the mix 4E-GH61 (2) and mix 4E-EG (3).

Fig. 2: The glucose produced in Example 2, 1 = Experiment 1: without aeration, 2 = Experiment 2: continuous aeration, 3 = Experiment 3: aeration starting after 72 hours until the end.

Fig. 3: The effect of the aeration time on the glucose produced during the enzymatic hydrolysis, - - = without aeration, ••• = the aeration between the hydrolysis time is 0 and 100 hours, - the aeration between the hydrolysis time is 0 and 7 hours, and ------------- = aeration between the hydrolysis time is 72 and 100 hours.

Fig. 4: The effect of the aeration time on the glucose produced during the enzymatic hydrolysis in experiment 1 (■ = the aeration between the hydrolysis time is 0 and 100 hours) and 2 (□ = the aeration between the hydrolysis time is 72 and 100 hours).

Fig. 5: The effect of the aeration time on the glucose produced during the enzymatic hydrolysis, - ■ - the aeration between the hydrolysis time is 72 and 100 hours, and - • - the aeration between the hydrolysis time is 0 and 7 hours.

Detailed description of the invention

Throughout this specification and the appended claims, the terms "comprise" and "include", and variations such as "comprise", "comprising", "includes" and "including", should be interpreted in a manner inclusive. In other words, these terms are intended to convey the possible inclusion of other elements or integers not specifically mentioned, when the context allows. The articles "a" and "a" are used herein to refer to one or more than one (ie, one or at least one) of the grammatical object of the article. As an example, "an item" can mean one item or more than one item.

In the context of the present invention, "improved", "increased", "reduced" are used to indicate that the present invention shows an advantage compared to the same situation, procedure or procedure conditions, except that no extra oxygen is added . Within the context of the present invention "measured under the same conditions" or "analyzed under the same conditions", etc., means that the process of the invention and the same procedure without (or with less) addition of oxygen are carried out in the same conditions (except for the addition of oxygen), and that the results of the present procedure, if compared with the procedure without (or with less) addition of oxygen, are measured using the same conditions, preferably using the same test and / or methodology, more preferably within the same or a parallel experiment. The hydrolysis conditions are an example of such conditions.

In the prior art it is suggested to improve the hydrolysis of the cellulolytic material using anaerobic conditions (document W02010 / 080407) or vacuum (document WO2009 / 046538) during enzymatic hydrolysis. In the procedures of both documents, the oxygen level was decreased. It has surprisingly been found that the hydrolysis of the present invention shows results in an improved reaction product that gives higher amounts of (reduced) sugar products and / or desired fermentation products in fermentation after hydrolysis compared to a procedure in which oxygen is not added. In general, an increase in glucose conversion of 5 to 15% w / w, or even up to 25% w / w is observed.

Oxygen can be added in various ways. For example, oxygen can be added as oxygen gas, oxygen-enriched gas, such as oxygen-enriched air, or air (an example of oxygen-containing gas). Oxygen can be added continuously or discontinuously. By the term "oxygen is added" is meant that oxygen is added to the liquid phase (comprising the lignocellulosic material) in the hydrolysis reactor, and not that oxygen is present in the headspace in the reactor above the liquid phase (in combination with slow or non-existent agitation), whereby oxygen has to diffuse from the headspace to the liquid phase. Thus, preferably, oxygen is added as bubbles, most preferably as small bubbles.

In the event that the enzyme can be damaged by the presence or addition of oxygen, a more moderate supply of oxygen can be used. In that case, a balance can be found between improved glucose production and enzyme behavior. The addition of oxygen to the cellulolytic material can be carried out during enzymatic hydrolysis. In the event that oxygen is added in gaseous form, an oxygen-containing gas can be introduced, for example blown, into the liquid contents of the cellulolytic material hydrolysis reactor. In another embodiment of the invention, the oxygen-containing gas is introduced into the liquid stream of cellulolytic material that will enter the hydrolysis reactor. In yet another embodiment of the invention, the oxygen-containing gas is introduced along with the cellulolytic material entering the hydrolysis reactor, or with part of the liquid reactor contents passing through an external loop of the reactor. In most cases, adding oxygen before entering the hydrolysis reactor is not enough, and adding oxygen can also be done during hydrolysis. In another embodiment of the invention, the gas phase present in the upper part of the reactor (headspace) is continuously or discontinuously renewed with the oxygen-containing gas. In the latter case, (vigorous) mixing or agitation is necessary to achieve that the oxygen in the form of bubbles and / or by diffusion reaches the liquid contents of the reactor, preferably in combination with overpressure in the reactor. In general, washing the headspace with air, in combination with (vigorous) mixing or stirring, can introduce sufficient oxygen into the cellulosic material in the hydrolysis reactor for reactors up to a size of 100 liters to 1 m3. According to the invention, the volume of the reactor is at least 1 m3. On a larger scale, for example in a reactor of 50 m3 or more, for example 100 m3, so much energy is needed for vigorous stirring that, economically, it will not be applied in a commercially operating process.

In accordance with the present invention, oxygen can be added during part of the hydrolysis step. The addition of oxygen during only part of the hydrolysis can be done, for example, in the event of oxidative damage to the enzyme or enzymes. In the event that the oxygen present in the contents of the hydrolysis reactor or the sugar product or hydrolyzate formed in the hydrolysis stage can influence or alter the subsequent fermentation stage, the addition of oxygen can be carried out, except during the last part of the hydrolysis, and thus (most of) the oxygen is consumed before the hydrolyzed biomass enters the fermentation reactor. Advantageously, oxygen, preferably in the form of bubbles (gaseous), is added in the last part of the hydrolysis step.

The inventors hypothesized that in the first part of the (enzymatic) hydrolysis (step) the amorphous polysaccharides hydrolyze to sugars, such as glucose, and that in the second part of the hydrolysis step the remaining crystalline polysaccharides are converted in sugars. Amorphous polysaccharides are converted for example to oligosaccharides by endoglucanases, after which the oligosaccharides can be converted by cellobiohydrolase and beta-glucosidase (BG) to sugars. According to the present hypothesis, amorphous polysaccharides are located on the outside of polysaccharides or polysaccharide complexes, whereas crystalline polysaccharides are located relatively more on the inside of polysaccharides or polysaccharide complexes present in lignocellulosic material. Thus, the conversion of the crystalline polysaccharides can continue even when most of the amorphous polypeptides are hydrolyzed. Especially, the addition of oxygen is beneficial during the hydrolysis of the crystalline polysaccharides, for example in the degradation of the polysaccharides into oligosaccharides. According to this hypothesis, the addition of oxygen is especially useful in the second part of the hydrolysis stage. In general, a shorter oxygen addition time (or a shorter second hydrolysis part) is required in the case of relatively low amounts of crystalline polysaccharides in the lignocellulosic material compared to the hydrolysis of a lignocellulosic material in which they are Relatively higher amounts of crystalline polysaccharides are present. The inventors have also argued that the addition of oxygen is beneficial for the hydrolysis of crystalline polysaccharides. Therefore, the addition of oxygen is very useful, especially in the phase in which the crystalline polysaccharides are attacked by the enzymes. Outside of this phase, not adding oxygen could be more effective. Therefore, the oxygen supply can start only in the second or second half of the hydrolysis. At the end of the hydrolysis, when most of the crystalline polysaccharides have degraded, the addition of oxygen is preferably stopped. In the latter part of the second part or second half of the hydrolysis, most of the polysaccharides are converted to oligosaccharides which, during a further breakdown into smaller sugars, no longer need oxygen. Therefore, at the end of the hydrolysis process, preferably less oxygen is added to the lignocellulosic material, compared to adding oxygen during the aerated part of the time, or more preferably, at the end of the hydrolysis process, no oxygen is added to the lignocellulosic material. This hypothesis is only given as a possible explanation of the effect observed by the inventors, and the present invention does not confirm or deny the accuracy of this theory.

The inventors have also observed that aeration during an enzymatic hydrolysis procedure at the start of the hydrolysis procedure results in increased glucose production during hydrolysis.

The effect of aeration is shown in Figure 3. Compared to non-aerated hydrolysis (shown as a "not aerated" curve), aeration at the start of the hydrolysis procedure (shown as a "0-7 hour" aeration curve) will result in an immediate increase in glucose production, and for example, already after 24 hours of hydrolysis you will find a glucose production corresponding to a glucose production without aeration of 60 hours of hydrolysis under identical conditions (except for aeration). Compared to non-aerated hydrolysis, aeration of the last part of the hydrolysis procedure (shown as "aeration 72-100 hour" curve) will result in an immediate increase in glucose production after aeration, and for example Already after 24 hours after the start of aeration (at 72 hours) in the hydrolysis procedure, a 30% increase in glucose production will be found compared to glucose production without aeration under identical conditions (except by aeration). The inventors believe that using aeration at the beginning, as well as in the last part, of the hydrolysis procedure (with a period without aeration between aeration intervals) could increase glucose production, thereby resulting in increased glucose production that is greater than each of the two increases separately. The present explanation is given to guide and instruct the person skilled in the art to select the appropriate conditions for the present hydrolysis procedure.

In the Examples several examples of partial aeration during the enzymatic hydrolysis procedure are given, to show the beneficial effect of the present invention. This beneficial effect is found for various substrates or raw materials, and is therefore believed to be present for the hydrolysis of all kinds of substrates or raw materials.

In the Examples several examples of enzyme compositions for the enzymatic hydrolysis process are given, to show the beneficial effect of the present invention. This beneficial effect is found for various enzyme compositions and is therefore believed to be present for all kinds of hydrolysis enzyme compositions.

According to a preferred embodiment of the invention, the part of the time in which less or preferably no oxygen is added is 10 to 80%, preferably 20 to 80%, more preferably 30 to 80% and, most preferably, 40 80% of the time of total enzymatic hydrolysis. According to a further preferred embodiment of the invention, the part of the time in which more oxygen is added is 8 to 50% and, most preferably, 10 to 50% of the time of total enzymatic hydrolysis. According to the invention, the concentration of oxygen in the liquid phase during the part of time in which oxygen is added is at least 2 times, preferably at least 4 times, more preferably at least 10 times the concentration of oxygen in the phase Liquid during the part of the time when less or no oxygen is added.

In a further preferred embodiment of the invention, during the part of the time in which the addition of oxygen in the liquid phase takes place (by aeration or addition of oxygen), the concentration of oxygen (DO) in the liquid phase in which lignocellulosic material is present during enzymatic hydrolysis, it is at least 0.001 mol / m3, preferably at least 0.002 mol / m3, more preferably at least 0.003 mol / m3, and even more preferably more than 0.01 mol / m3, for example more than 0.02 mol / m3 or 0.03 mol / m3. In reactors of less than 1 m3, DO values below 0.01 mol / m3 or 0.02 mol / m3 will be obtained by slow stirring. Vigorous mixing or stirring on such a scale introduces part of the gaseous phase of the headspace into the reaction liquid. For example, mixing or stirring can create a whirlpool that directs oxygen into the liquid. In general, washing the headspace with air in combination with (vigorous) mixing or agitation will introduce sufficient oxygen into the cellulosic material in the hydrolysis reactor for reactors up to a size of 100 liters to 1 m3. According to the invention, the volume of the reactor is at least 1 m3. On a larger scale, for example in a reactor of 50 m3 or more, for example 100 m3, so much energy is needed for vigorous stirring that from an economic point of view this will not apply in a commercially operating process. In general, in larger reactors, agitation or mixing without introduction of air or oxygen will result in OD values of less than 0.01 mol / m3.

In yet another preferred embodiment of the invention, during the generation or production of oxygen, the concentration of oxygen in the liquid phase (aeration or addition of oxygen), the concentration of oxygen in the liquid phase in which the lignocellulosic material is present during enzymatic hydrolysis is during the part of the time in which oxygen is preferably added at most 80% of the saturation concentration of oxygen under the conditions of the hydrolysis reaction, more preferably at most 0.12 mol / m3, even more preferably at most 0.09 mol / m3, even more preferably at most 0.06 mol / m3, even more preferably at most 0.045 mol / m3, and most preferably at most 0.03 mol / m3. Temperature and pressure will influence DO. The preferred and exemplary mol / m3 values given above refer to normal atmospheric pressure and a temperature of about 62 ° C. One skilled in the art will appreciate favorable OD values based on the present teachings.

In a further preferred embodiment of the invention, the concentration of oxygen in the liquid phase, in which the lignocellulosic material is present during enzymatic hydrolysis, is during the part of time in which the least or No oxygen is added less than 0.02 mol / m3, preferably less than 0.01 mol / m3, more preferably less than 0.005 mol / m3, and most preferably less than 0.001 mol / m3.

The addition of oxygen in the form of air or other oxygen-containing gas according to the invention can also be used to stir or at least partially mix the contents of the hydrolysis reactor. The present process of the invention shows advantages, especially on a pilot plant scale and an industrial scale. As noted above, the hydrolysis reactor has a volume of 1m3 or more, preferably more than 10m3, and more preferably 50m3 or more. In general, the hydrolysis reactor will be smaller than 3,000 m3 or 5,000 m3. The inventors raise the theory that, especially on a larger scale, insufficient oxygen is available for hydrolysis, which could be due to limitations in the transfer of oxygen in the reactor, for example in cellulolytic biomass. In laboratory-scale experiments, this oxygen deficiency may play a less important role. The ratio of the surface area (or oxygen contact area of the reactor contents) to the reactor volume is more favorable for small-scale experiments than large-scale experiments. Also, mixing in small-scale experiments is relatively easier than large-scale. During these small-scale experiments, oxygen transport from the hydrolysis reactor headspace is also faster than compared to the situation in larger-scale experiments. This theory only gives a possible explanation of the effect observed by the inventors, and the present invention does not confirm or deny the accuracy of this theory. In accordance with a further embodiment of the invention, the addition of oxygen can be used to at least partially control the hydrolysis procedure.

The process of the invention is advantageously applied in combination with the use of thermostable enzymes.

A "thermostable" enzyme means that the enzyme has an optimum temperature of 60 ° C or higher, for example 70 ° C or higher, such as 75 ° C or higher, for example 80 ° C or higher, such as 85 ° C or higher. For example, they can be isolated from thermophilic microorganisms or they can be designed by the person skilled in the art and artificially synthesized. In one embodiment, the polynucleotides can be isolated or obtained from thermophilic or thermotolerant filamentous fungi, or isolated from non-thermophilic or non-thermotolerant fungi but known to be thermostable.

By "thermophilic fungus" is meant a fungus that grows at a temperature of 50 ° C or higher. By "thermotolerant fungus" is meant a fungus that grows at a temperature of 45 ° C or higher, with a maximum close to 50 ° C.

Examples of thermophilic fungal strains are Rasamsonia emersonii (formerly known as Talaromyces emersonii; Talaromyces emersonii, Penicillium geosmithia emersonii, and Rasamsonia emersonii are used interchangeably herein).

Suitable thermophilic or thermotolerant fungal cells may be a Humicola, Rhizomucor, Myceliophthora, Rasamsonia, Talaromyces, Thermomyces, Thermoascus or Thielavia cell , preferably a Rasamsonia emersonii cell . The preferred thermophilic or thermotolerant fungi are Humicola grísea var. thermoidea, Humicola, Myceliophthora thermophila, Papulaspora thermophilia, Rasamsonia byssochlamydoides, Rasamsonia emersonii, Rasamsonia argillacea, Rasamsonia eburnean, brevistipitata Rasamsonia, Rasamsonia cylindrospora, Rhizomucor pusillus, Rhizomucor miehei, Talaromyces bacillisporus, leycettanus Talaromyces, Talaromyces thermophilus, Thermomyces lenuginosus, Thermoascus crustmaw, Thermoascus thermophilus Thermoascus aurantiacus and Thielavia terrestris.

Thermophilic fungi are not restricted to a specific taxonomic order, and occur throughout the fungal tree of life. Examples are Rhizomucor in Mucorales, Myceliophthora in Sordariales, and Talaromyces, Thermomyces and Thermoascus in Eurotiales (Mouchacca 1997). Most Talaromyces species are mesophilic, but the species in the Emersorii and Thermophila sections are exceptions . The Emersonii section includes Talaromyces emersonii, Talaromyces byssochlamydoides, Talaromyces bacillisporus and Talaromyces leycettanus, all of which grow well at 40 ° C.

Talaromyces bacillisporus is thermotolerant, T. leycettanus is thermotolerant to thermophilic, and T. emersonii and T. byssochlamydoides are truly thermophiles (Stolk and Samson 1972). The only member of the Thermophila section of Talaromyces, T. thermophilus, grows rapidly at 50 ° C (Evans and Stolk 1971; Evans 1971; Stolk and Samson 1972). The current classification of these thermophilic Talaromyces species is mainly based on phenotypic and physiological characters, such as their ability to grow above 40 ° C, the color of the ascospores, the structure of the ascornatal cover, and the formation of a certain type of anamorphosis. Stolk and Samson (1972) established that members of the Emersonii section have anamorphs of either Paecilomyces ( T. byssochlamydoides and T. leycettanus) or the Penicillium cylindrosporum series ( T. emersonii and T. bacillisporus). Subsequently, Pitt (1979) transferred species belonging to the Penicillium cylindrosporum series to the genus Geosmithia, based on various characters such as the formation of conidia from terminal pores instead of collula (necks), a character of Penicillium and Paecilomyces. Within the genus Geosmithia, only G. argillacea is heat tolerant , and Stolk et al. (1969) and Evans (1971) proposed a connection with members of the Emersonii section of Talaromyces. The phylogenetic relationship of the thermophilic Talaromyces species within Talaromyces and the Trichocomaceae is unknown. See J. Houbraken, Antonie van Leeuwenhoek 2012 Feb; 101 (2): 403-21.

Rasamsonia is a new genus comprising the thermotolerant and thermophilic Talaromyces and Geosmithia species (J. Houbraken et al, see above). Based on phenotypic, physiological, and molecular data, Houbraken et al. they proposed to transfer the species T. emersonii, T. byssochlamydoides, T. eburneus, G. argillacea and G. cylindrospora to Rasamsonia gen. Nov. Talaromyces emersonii, Penicillium geosmithia emersonii and Rasamsonia emersonii are used interchangeably here.

Preferred thermophilic fungi are Rasamsonia byssochlamydoides, Rasamsonia emersonii, Thermomyces lenuginosus, Talaromyces thermophilus, Thermoascus crustaceus, Thermoascus thermophilus, and Thermoascus aurantiacus.

"Filamentous fungi" includes all filamentous forms of the Eumycota and Oomycota subdivision (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) . Filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is due to elongation of the hyphae, and carbon catabolism is necessarily aerobic. Filamentous fungal strains include, but are not limited to, Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Geosmithia, Humicola, Penicill, Neocall, Neocall, Neocall , Panerochaete, Pleurotus, Rasamsonia, Schizophyllum, Talaromyces, Thermoascus, Thermomyces, Thielavia, Tolypocladium, and Trichoderma.

Various strains of filamentous fungi are easily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL). Examples of such strains include Aspergillus niger CBS 513.88, Aspergillus oryzae ATCC 20423, IFO 4177, ATCC 1011, ATCC 9576, ATCC14488-14491, ATCC 11601, ATCC12892, P. chrysogenum CBS 455.95, Penicillium citrinum ATCC 38065, PenCCillis ATCC 38065, Penicillium citrinum ATCC 38065, Penicillium citrinum ATCC 38065, Penicillium citrinum ATCC 38065, Penicillium citrinum ATCC 38065, Penis emersonii CBS 393.64, Acremonium chrysogenum ATCC 36225 or ATCC 48272, Trichoderma reesei ^{ATCC 26921 or ATCC 56765 or} ATcC ^{26921, Aspergillus sojae ATCC11906, Chrysosporium lucknowense C1, Garg} 27K, VKM F-3500-D.

An advantage of expression and production of enzymes (eg, at least two, three, or four different cellulases) in a suitable microorganism may be a high yield of enzyme composition that can be used in the method of the present invention.

According to the invention, by adding oxygen it is possible to achieve many process advantages, including optimal temperature conditions, reduced process time, reduced enzyme dosing, enzyme reuse and other process optimizations, resulting in reduced costs. . Advantageously, the invention provides a process in which the hydrolysis step is carried out under improved conditions. The invention also provides a process involving hydrolysis that has a reduced process time. Additionally, the invention provides a process, in which the enzyme dosage can be reduced and at the same time the production of a useful hydrolysis product is kept at the same level. Another advantage of the invention is that the present process involving hydrolysis can result in process conditions that are optimized. A further advantage of the invention is that the production of a useful hydrolysis product from the process involving hydrolysis is increased using the same enzyme dosage.

Stable enzyme composition

Stable enzyme composition here means that the enzyme composition retains its activity after 30 hours of hydrolysis reaction time, preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70 %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of its initial activity after 30 hours of hydrolysis reaction time. Preferably, the enzyme composition retains activity after 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 hours of hydrolysis reaction time.

The enzyme composition can be prepared by fermentation of a suitable substrate with a suitable microorganism, for example Rasamsonia emersonii or Aspergillus niger, in which the microorganism produces the enzyme composition. The microorganism can be altered to improve or create the cellulase composition. For example, the microorganism can mutate by classical strain improvement procedures or by recombinant DNA techniques. Therefore, the microorganisms mentioned here can be used as such to produce the cellulase composition, or they can be altered to increase production or produce an altered cellulase composition that could include heterologous cellulases, and therefore enzymes that are not originally produced by that microorganism. Preferably a fungus, more preferably a filamentous fungus, is used to produce the cellulase composition. Advantageously, a thermophilic or thermotolerant microorganism is used. Optionally, a substrate that induces expression of the enzymes in the enzyme composition is used during the production of the enzyme composition.

The enzyme composition is used to release sugars from lignocellulose, which comprises polysaccharides. The main polysaccharides are cellulose (glucans), hemicelluloses (xylans, heteroxylanes, and xyloglucans). Also, something Hemicellulose may be present in the form of glucomannans, for example in raw materials derived from wood. The enzymatic hydrolysis of these polysaccharides to soluble sugars, including both monomers and multimers, for example glucose, cellobiose, xylose, arabinose, galactose, fructose, mannose, rhamnose, ribose, galacturonic acid, glucuronic acid and other hexoses and pentoses occurs under the action of different enzymes that act in concert. By sugar product is meant the enzymatic hydrolysis product of the raw material or lignocellulosic material. The sugar product will comprise soluble sugars, including both monomers and multimers, preferably it will comprise glucose. Examples of other sugars are cellobiose, xylose, arabinose, galactose, fructose, mannose, rhamnose, ribose, galacturonic acid, glucuronic acid, and other hexoses and pentoses. The sugar product can be used as such or can be further processed, eg purified.

Furthermore, pectins and other pectic substances such as arabinan can make up a considerable proportion of the dry matter of typically cell walls from non-wood plant tissues (about one quarter to one half of dry matter may be pectin).

Cellulose is a linear polysaccharide composed of glucose residues linked by b-1,4 bonds. The linear nature of cellulose fibers and the stoichiometry of the bound glucose using b (aa respect) generates structures more susceptible to hydrogen bonding intermixed with the structures joined by a highly branched starch. In this way, cellulose polymers are generally less soluble, and form bonded fibers more tightly than the fibers found in starch.

Enzymes that can be included in the stable enzyme composition used in the invention are now described in more detail:

GH61, endoglucanases (EG) and exo-cellobiohydrolases (CBH) catalyze the hydrolysis of insoluble cellulose to products such as cellooligosaccharides (cellobiose as the main product), while the b-glucosidases (BG) convert the oligosaccharides, mainly cellobiose and celotriose, into glucose.

Hemicellulose is a complex polymer, and its composition often varies widely from one organism to another and from one tissue type to another. In general, a major component of hemicellulose is b-1,4-linked xylose, a five-carbon sugar. However, this xylose is often branched at 0 to 3 and / or 0 to 2 xylose atoms, and may be substituted with linkages to arabinose, galactose, mannose, glucuronic acid, galacturonic acid, or by esterification to acetic acid (and esterification of ferulic acid to arabinose). Hemicellulose may also contain glucan, which is a general term for b-linked six-carbon sugars (such as the b- (1,3) (1,4) glucans and heteroglycans mentioned above), and additionally glucomannans (in that both glucose and mannose are present in the linear main chain, linked together by links b).

Xylanases, together with other added enzymes, for example α-L-arabinofuranosidases, feruloyl and acetylxylan esterases, glucuronidases and β-xylosidases), catalyze the hydrolysis of hemicelluloses.

Pectic substances include pectins, arabinans, galactans, and arabinogalactans. Pectins are the most complex polysaccharides in the plant cell wall. They accumulate around a central chain of D-galacturonic acid units attached to (1,4) intermingled to some degree with L-rhamnose. In any cell wall, there are a number of structural units that fit this description, and it has generally been considered that, in a single pectic molecule, the central chains of different structural units are continuous with each other.

The main types of structural unit are: galacturonan (homogalacturonan), which may be substituted with methanol in the carboxyl group and acetate in O-2 and O-3; rhamnogalacturonan I (RGI), in which the galacturonic acid units alternate with rhamnose units carrying side chains of bound galactane (1,4) and bound arabinan (1,5). The arabinan side chains can be attached directly or indirectly to rhamnose through the galactane chains; xylogalacturonan, with individual xylosyl units in galacturonic acid O-3 (closely related to RGI); and rhamnogalacturonan II (RGII), a particularly complex minority unit containing unusual sugars, for example apiosa. A RGII unit may contain two apiosyl residues which, under suitable ionic conditions, can reversibly form borate esters.

A composition for use in a method of the invention comprises at least two different cellulases, one of which is GH61, and preferably at least one hemicellulase. A composition for use in the invention may not comprise xylanases. Furthermore, a composition of the invention may comprise auxiliary enzymatic activity, that is, additional activity that, either directly or indirectly, leads to degradation of lignocellulose. Examples of such ancillary activities are mentioned here.

Thus, a composition for use in the invention comprises GH61, and can comprise endoglucanase activity and / or cellobiohydrolase activity and / or b-glucosidase activity. For example, a composition for use in the invention may additionally comprise two endoglucanase activities, for example endo-1,3 (1,4) -b glucanase activity and endo-b-1,4-glucanase activity. Such a composition may also comprise one or more xylanase activities. Such a composition may comprise an auxiliary enzyme activity.

A composition for use in the invention, as defined in the claims, can be derived from Rasamsonia emersonii. In the invention, it is anticipated that a core set of enzymatic activities (lignocellulose degradants ) may be derived from Rasamsonia emersonii. Rasamsonia emersonii can provide a very effective set of activities as demonstrated here for the hydrolysis of lignocellulosic biomass. This activity can then be supplemented by additional enzyme activities from other sources. Such additional activities can be derived from classical sources and / or produced by a genetically modified organism.

The activities in a composition for use in the invention can be thermostable. Here, this means that the activity has a temperature optimum of about 602C or higher, for example about 70 ° C or higher, such as about 75 ° C or higher, for example about 80 ° C or higher, such as 85 ° C or older. Activities in a composition for use in the invention typically will not have the same temperature optima, but preferably will nonetheless be thermoset.

Furthermore, the enzymatic activities in a composition for use in the invention may be able to work at low pH. For the purposes of this invention, low pH indicates a pH of about 5.5 or less, about 5 or less, about 4.9 or less, about 4.8 or less, about 4.7 or less, about 4.6 or less, about 4.5 or less, about 4.4 or less, about 4.3 or less, about 4.2 or less, about 4.1 or less, about 4.0 or less, about 3.9 or lesser, or about 3.8 or less, about 3.7 or less, about 3.6 or less, or about 3.5 or less.

The activities in a composition for use in the invention can be defined by a combination of any of the above pH and temperature optimum values.

The composition used in a method of the invention may comprise, in addition to activities derived from Rasamsonia , a cellulase (eg, a derivative from a source other than Rasamsonia ) and / or a hemicellulase (eg, a derivative from a different source). Rasamsonia ) and / or a pectinase.

A composition for use in the invention comprises at least two cellulases; it can comprise one, two, three, four or more classes of cellulase, for example one, two, three or four or all of a GH61, an endoglucanase (EG), one or two exo-cellobiohydrolase (CBH) and a p-glucosidase (BG). A composition for use in the invention may comprise two or more of any of these classes of cellulase.

A composition for use in the invention may comprise an activity having a different type of cellulase activity and / or hemicellulase activity and / or pectinase activity than that provided by the composition for use in a method of the invention. For example, a composition for use in the invention may comprise one type of cellulase and / or hemicellulase activity and / or pectinase activity provided by a composition as described herein, and a second type of cellulase and / or hemicellulase and / or pectinase activity provided by an additional cellulase / hemicellulase / pectinase.

Here, a cellulase is any polypeptide that is capable of degrading or modifying cellulose. A polypeptide that is capable of degrading cellulose is one that is capable of catalyzing the process of degrading cellulose in smaller units either partially, for example cellodextrins, or entirely in glucose monomers. A cellulase for use in the invention can give rise to a mixed population of cellodextrins and glucose monomers when contacted with the cellulase. Such degradation will typically take place through a hydrolysis reaction.

GH61 proteins (family 61 of glucoside hydrolases, or sometimes called EGIV) are oxygen-dependent polysaccharide monooxygenases (PMO) according to the most recent literature. These proteins are often mentioned in the literature as enhancing the action of cellulases on lignocellulose substrates. GH61 was originally classified as an endoglucanase based on the measurement of very weak endo-1,4-p-d-glucanase activity in a family member. The term "GH61", as used herein, should be understood as a family of enzymes that share conserved sequence portions and common folds in order to classify them into family 61 of the well established CAZY GH classification system (http: //www.cazy .org / GH61 .html). The 61 family of glycoside hydrolases is a member of the EC 3.2.1 family of glycoside hydrolases. GH61 is used here as part of cellulases.

Here, a hemicellulase is any polypeptide that is capable of degrading or modifying hemicellulose. That is, a hemicellulase may be capable of degrading or modifying one or more of xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan. A polypeptide that is capable of degrading a hemicellulose is one that is capable of catalyzing the breakdown process of hemicellulose into smaller polysaccharides, either partially, for example in oligosaccharides, or completely, in sugar monomers, for example sugar monomers hexose or pentose. A hemicellulase for use in the invention can give rise to a mixed population of oligosaccharides and sugar monomers when contacted with the hemicellulase. Such degradation will typically take place through a hydrolysis reaction.

Here, a pectinase is any polypeptide that is capable of degrading or modifying pectin. A polypeptide that is capable of degrading pectin is one that is capable of catalyzing the pectin cleavage procedure in larger units. small, either partially, for example in oligosaccharides, or completely in sugar monomers. A pectinase for use in the invention can give rise to a mixed population of oligosaccharides and sugar monomers when contacted with the pectinase. Such degradation will typically take place through a hydrolysis reaction.

Accordingly, a composition for use in the invention may comprise any cellulase, but at least one GH61, one cellobiohydrolase, one endo-p-1,4-glucanase, one p-glucosidase or one b (1,3) (1, 4) -glucanase.

Here, a cellobiohydrolase (EC 3.2.1.91) is any polypeptide that is capable of catalyzing the hydrolysis of 1,4-p-D-glucosidic linkages into cellulose or celotetraose, releasing cellobiose from the ends of the chains. This enzyme can also be called cellulase 1,4-p-cellobiosidase, 1,4-p-cellobiohydrolase, 1,4-p-D-glucan cellobiohydrolase, avicelase, exo-1,4-p-D-glucanase, exocelobiohydrolase or exoglucanase.

Here, an endo-p-1,4-glucanase (EC 3.2.1.4) is any polypeptide that is capable of catalyzing the endohydrolysis of 1,4-p-D-glucosidic linkages in cellulose, lichenine or cereal p-D-glucans. Such a polypeptide may also be capable of hydrolyzing 1,4-linkages into p-D-glucans that also contain 1,3-linkages. This enzyme can be called cellulase, avicelase, p-1,4-endoglucan hydrolase, p-1,4-glucanase, carboxymethyl cellulase, celludextrinase, endo-1,4-pD-glucanase, endo-1,4-pD-glucanohydrolase, endo -1,4-p-glucanase or endoglucanase.

Here, a p-glucosidase (EC 3.2.1.21) is any polypeptide that is capable of catalyzing the hydrolysis of terminal non-reducing p-D-glucose residues, with release of p-D-glucose. Such a polypeptide can have broad specificity for p-D-glucosides, and can also hydrolyze one or more of the following: a p-D-galactoside, an a-L-arabinoside, a p-D-xyloside, or a p-D-fucoside. This enzyme can also be called amygdalase, p-D-glucoside glucohydrolase, cellobiase or gentobiase.

Here, a p- (1,3) (1,4) -glucanase (EC 3.2.1.73) is any polypeptide that is capable of catalyzing the hydrolysis of 1,4-pD-glycosidic linkages in pD-glucans containing links 1,3 and 1,4. Such a polypeptide can act on the lichenine and cereal p-D-glucans, but not on the p-D-glucans that contain only 1,3- and 1,4 linkages. This enzyme may also be referred to as licheninase, 1,3-1,4-p-D-glucan 4-glucanohydrolase, p-glucanase, endo-p-1,3-1,4-glucanase, lichenase, or mixed-binding p-glucanase. An alternative for this type of enzyme is EC 3.2.1.6, which is described as endo-1,3 (4) -beta-glucanase. This type of enzyme hydrolyzes 1,3 or 1,4 linkages into beta-D-glucanase when the glucose moiety whose reducing group is involved in the link to be hydrolyzed is itself substituted at C-3. Alternative names include endo-1,3-beta-glucanase, laminarinase, 1.3- (1,3; 1,4) -beta-D-glucan 3 (4) glucanohydrolase; substrates include beta-D-glucans from laminarin, lichen and cereal.

A composition for use in the invention may comprise any hemicellulase, for example an endoxylanase, a p-xylosidase, an aL-arabionofuranosidase, aD-glucuronidase, an acetyl xylan esterase, a feruloyl esterase, a coumaroyl esterase, a-galactosidase, a p-galactosidase, a p-mannanase, or a p-mannosidase.

Here, an endoxylanase (EC 3.2.1.8) is any polypeptide that is capable of catalyzing the endohydrolysis of the 1,4-p-D-xylosidic linkages in xylan. This enzyme may also be called endo-1,4-p-xylanase or 1,4-p-D-xylan xylanhydrolase. An alternative is EC 3.2.1.136, a glucuronoarabinoxylan endoxylanase, an enzyme that is capable of hydrolyzing 1,4 xylosidic linkages in glucuronoarabinoxylan.

Here, a p-xylosidase (EC 3.2.1.37) is any polypeptide that is capable of catalyzing the hydrolysis of 1,4-p-D-xylanes, to remove successive D-xylose residues from the non-reducing ends. Such enzymes can also hydrolyze xylobiose. This enzyme can also be called xylan 1,4-p-xylosidase, 1,4-p-D-xylan xylohydrolase, exo-1,4-p-xylosidase or xylobiase.

Here, an aL-arabinofuranosidase (EC 3.2.1.55) is any polypeptide that is capable of acting on aL-arabinofuranosides, aL-arabinan containing junctions (1,2) and / or (1,3) and / or (1, 5), arabinoxylans and arabinogalactans. This enzyme can also be called a-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

Here, an α-D-glucuronidase (EC 3.2.1.139) is any polypeptide that is capable of catalyzing a reaction in the following way: alpha-D-glucuronoside H (2) O = a D-glucuronate alcohol. This enzyme can also be called alpha-glucuronidase or alpha-glucosiduronase. These enzymes can also hydrolyze 4-O-methylated glucuronic acid which may also be present as a substituent on xylans. The alternative is EC 3.2.1.131: alpha-1,2-glucuronosidase xylan, which catalyzes the hydrolysis of alpha-1,2- (4-O-methyl) glucuronosyl linkages.

Here, an acetyl xylan esterase (EC 3.1.1.72) is any polypeptide that is capable of catalyzing the deacetylation of xylan and xylo-oligosaccharides. Such a polypeptide can catalyze the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-naphthyl acetate or p-nitrophenyl acetate, but typically not from triacetylglycerol. Such a polypeptide typically does not act on acetylated mannan or pectin.

Here, a feruloyl esterase (EC 3.1.1.73) is any polypeptide that is capable of catalyzing a reaction of the form: feruloyl-saccharide H2O = ferulate saccharide. The saccharide can be, for example, an oligosaccharide or a polysaccharide. Typically it can catalyze the hydrolysis of the 4-hydroxy-3-methoxycinmoyl (feruloyl) group from an esterified sugar, which is typically arabinose on "natural" substrates. P-Nitrophenol acetate and methyl ferulate are typically poorer substrates. This enzyme may also be referred to as cinnamoyl ester hydrolase, ferulic acid esterase, or hydroxycinmoyl esterase. It can also be called a hemicellulase helper enzyme, since it can help xylanases and pectinases to break hemicellulose and pectin from the plant cell wall.

Here, a coumaroyl esterase (EC 3.1.1.73) is any polypeptide that is capable of catalyzing a reaction of the form: coumaroyl saccharide H2O = cumarate saccharide.

The saccharide can be, for example, an oligosaccharide or a polysaccharide. This enzyme may also be referred to as trans-4-coumaroyl esterase, trans-p-coumaroyl esterase, p-coumaroyl esterase, or p-coumaric acid esterase. This enzyme is also included within EC 3.1.1.73, so it can also be called feruloyl esterase.

Here, an α-galactosidase (EC 3.2.1.22) is any polypeptide that is capable of catalyzing the hydrolysis of terminal, non-reducing α-D-galactose residues in α-D-galactosides, including galactose, galactomannans, galactanes, and arabinogalactans. Such a polypeptide may also be capable of hydrolyzing α-D-fucosides. This enzyme can also be called melibiasa.

Here, a p-galactosidase (EC 3.2.1.23) is any polypeptide that is capable of catalyzing the hydrolysis of terminal non-reducing b-D-galactose residues in p-D-galactosides. Such a polypeptide may also be capable of hydrolyzing α-L-arabinosides. This enzyme can also be called exo- (1®4) -p-D-galactanase or lactase.

Here, a p-mannanase (EC 3.2.1.78) is any polypeptide that is capable of catalyzing the random hydrolysis of 1,4-p-D-mannosidic linkages in mannans, galactomannans, and glucomannans. This enzyme may also be referred to as mannan endo-1,4-p-mannosidase or endo-1,4-mannanase.

Here, a p-mannosidase (EC 3.2.1.25) is any polypeptide that is capable of catalyzing the hydrolysis of terminal non-reducing p-D-mannose residues into p-D-mannosides. This enzyme can also be called mannanase or manase.

A composition for use in the invention may comprise any pectinase, for example an endo polygalacturonase, a pectin methyl esterase, an endogalactanase, a beta galactosidase, a peptin acetyl esterase, an endopectin lyase, pectate lyase, alpha rhamnosidase, a exo-galacturonase, an expoligalacturonate lyase, a ramnogalacturonan hydrolase, a ramnogalacturonan lyase, a ramnogalacturonan acetyl esterase, a rhamnogalacturonan galacturonohydrolase, a xylogalacturonase.

Here, an endo-polygalacturonase (EC 3.2.1.15) is any polypeptide that is capable of catalyzing the random hydrolysis of 1,4-a-D-galactosiduronic bonds in pectate and other galacturonans. This enzyme may also be referred to as polygalacturonase, pectin depolymerase, pectinase, endopolygalacturonase, pectolase, pectin hydrolase, pectin polygalacturonase, poly-a-1,4-galacturonide glucanohydrolase, endogalacturonase; endo-D-galacturonase or poly (1,4-a-D-galacturonide) glucanohydrolase.

Here, a pectin methyl esterase (EC 3.1.1.11) is any enzyme that is capable of catalyzing the reaction: pectin n H2O = n methanol pectate. The enzyme may also be known as pectinesterase, pectin demethoxylase, pectin methoxylase, pectin methyl esterase, pectase, pectin esterase, or pectin pectylhydrolase.

Here, an endo-galactanase (EC 3.2.1.89) is any enzyme capable of catalyzing the endohydrolysis of 1,4-p-D-galactosidic bonds in arabinogalactans. The enzyme may also be known as arabinogalactan endo-1,4-p-galactosidase, endo-1,4-p-galactanase, galactanase, arabinogalactanase or arabinogalactan 4-p-D-galactanohydrolase.

Here, a pectin acetyl esterase is defined herein as any enzyme having an acetyl esterase activity that catalyzes the deacetylation of acetyl groups on the hydroxyl groups of the GalUA residues of the pectin

Here, an endo-pectin lyase (EC 4.2.2.10) is any enzyme capable of catalyzing the eliminatory cleavage of (1®4) -aD-galacturonan methyl ester to give oligosaccharides with 4-deoxy-6-O-methyl-aD groups. -galact-4-enuronosil at its non-reducing ends. The enzyme may also be known as pectin lyase, pectin frans-eliminase; endo-pectin lyase, polymethylgalacturonic transeliminase, pectin methyltranseliminase, pectoliase, PL, PNL or PMGL or (1®4) -6-O-methyl-a-D-galacturonanase lyase.

Here, a pectate lyase (EC 4.2.2.2) is any enzyme capable of catalyzing the eliminatory cleavage of (1®4) -aD-galacturonan to give oligosaccharides with 4-deoxy-aD-galact-4-enuronosyl groups at their non-ends reducers. The enzyme may also be known as polygalacturonic transeliminase, pectic acid transeliminase, polygalacturonate lyase, endopectin methyltranseliminase, pectate transeliminase, endogalacturonate transeliminase, pectic acid lyase, pectose lyase, α-1,4-D-endogalagase acid lyase, PPase-N, endo-a-1,4-polygalacturonic acid lyase, polygalacturonic acid lyase, pectin frans-eliminase, polygalacturonic acid frans-eliminase or (1®4) -aD-galacturonnan lyase.

Here, an alpha-rhamnosidase (EC 3.2.1.40) is any polypeptide that is capable of catalyzing the hydrolysis of terminal non-reducing a-L-rhamnose residues in a-L-rhamnosides or, alternatively, in rhamnogalacturonans. This enzyme may also be known as α-L-ramnosidase T, α-L-ramnosidase N or α-L-rhamnoside ramnohydrolase.

Here, exo-galacturonase (EC 3.2.1.82) is any polypeptide capable of hydrolyzing pectic acid from the non-reducing end, releasing digalacturonate. The enzyme can also be known as exo-poly-a-galacturonosidase, exopoligalacturonosidase or exopoligalacturanosidase.

Here, exo-galacturonase (EC 3.2.1.67) is any polypeptide capable of catalyzing: (1,4-aD-galacturonide) ⁿ + H ² O = (1,4-aD-galacturonide) ^n-1 + D-galacturonate . The enzyme may also be known as galactane 1.4-a-galacturonidase, exopoligalacturonase, poly (galacturonate) hydrolase, exo-D-galacturonase, exo-D-galacturonanase, exopoli-D-galacturonase, or poly (1,4-aD-galacturonide) galacturonohydrolase .

Here, the exopoligalacturonate lyase (EC 4.2.2.9) is any polypeptide capable of catalyzing the knockout cleavage of 4- (4-deoxy-aD-galact-4-enuronosyl) -D-galacturonate from the reducing end of pectate, i.e., pectin deesterified. This enzyme can be known as pectate disaccharide-lyase, pectate exo-lyase, exopectic acid transeliminase, exopectate lyase, exopolygalacturonic acid frans-eliminase, PATE, exo-PATE, exo-PGL or reducing end-disaccharide lyase (1 ® 4) -aD-galacturonan.

Here, rhamnogalacturonan hydrolase is any polypeptide that is capable of hydrolyzing the junction between galactosyluronic acid and rhamnopyranosyl in an endo manner in strictly alternate ramnogalacturonan structures, consisting of disaccharide [acid (1,2-alpha-L-ramnoyl- (1 , 4) -alpha-galactosyluronic].

Here, ramnogalacturonan lyase is any polypeptide that is any polypeptide that is capable of cleaving the α-L-Rhap- (1®4) -a-D-GalpA junctions in an endo-ramnogalacturonan manner by beta-deletion.

Here, ramnogalacturonan acetyl esterase is any polypeptide that catalyzes the deacetylation of the main chain of alternate residues of rhamnose and galacturonic acid in ramnogalacturonan.

Here, ramnogalacturonan galacturonohydrolase is any polypeptide that is capable of hydrolyzing galacturonic acid from the non-reducing end of strictly alternate ramnogalacturonan structures in an exo manner.

Here, xylogalacturonase is any polypeptide that acts on xylogalacturonan by cleavage of the p-xylose-substituted galacturonic acid backbone in an endo manner . This enzyme can also be known as xylogalacturonan hydrolase.

Here, an aL-arabinofuranosidase (EC 3.2.1.55) is any polypeptide that is capable of acting on aL-arabinofuranosides, aL-arabinan containing junctions (1,2) and / or (1,3) and / or (1, 5), arabinoxylans and arabinogalactans. This enzyme can also be called a-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

Here, endo-arabinanase (EC 3.2.1.99) is any polypeptide that is capable of catalyzing the endohydrolysis of 1,5-a-arabinofuranosidic junctions into 1,5-arabinan. The enzyme may also be known as endo-arabinase, arabinan endo-1,5-a-L-arabinosidase, endo-1,5-a-L-arabinanase, endo-a-1,5-arabanase; endo-arabanase or 1.5-a-L-arabinan 1,5-a-L-arabinanohydrolase.

A composition for use in the invention comprises at least one cellulase and GH61, and can comprise at least one hemicellulase and / or at least one pectinase. A composition for use in the invention may comprise a cellobiohydrolase, an endoglucanase and / or a p-glucosidase. Such a composition may also comprise one or more hemicellulases and / or one or more pectinases.

In addition, one or more (eg two, three, four or all) of an amylase, a protease, a lipase, a ligninase, a hexosyltransferase, a glucuronidase or an expansin or a cellulose-induced protein or a cellulose-integrating protein or a similar protein may be present in a composition for use in the invention (these have been referred to as auxiliary activities above).

A "protease" includes enzymes that hydrolyze peptide bonds (peptidases), as well as enzymes that hydrolyze bonds between peptides and other residues such as sugars (glucopeptidases). Many proteases are characterized according to EC 3.4, and are suitable for use in the invention. Some specific types of proteases include cysteine proteases, which include pepsin, papain, and serine proteases, which include chymotrypsins, carboxypeptidases, and metalloendopeptidases.

A "lipase" includes enzymes that hydrolyze lipids, fatty acids, and acylglycerides, including phosphoglycerides, lipoproteins, diacylglycerols, and the like. In plants, lipids are used as structural components to limit water loss and pathogen infection. These lipids include waxes derived from fatty acids, as well as cutin and suberin.

A "ligninase" includes enzymes that can hydrolyze or break the structure of the lignin polymers. Lignin-disrupting enzymes include lignin peroxidases, manganese peroxidases, laccases, and feruloyl esterases, and other enzymes described in the art that are known to depolymerize or otherwise break lignin polymers. Also included are enzymes capable of hydrolyzing the bonds formed between hemicellulosic sugars (specifically arabinose) and lignin. Ligninases include, but are not limited to, the following group of enzymes: lignin peroxidases (EC 1.11.1.14), manganese peroxidases (EC 1.11.1.13), laccases (EC 1.10.3.2), and feruloyl esterases (EC 3.1.1.73).

A "hexosyltransferase" (2.4.1-) includes enzymes that are capable of catalyzing a transferase reaction, but that can also catalyze a hydrolysis reaction, for example of cellulose and / or cellulose degradation products. An example of a hexosyltransferase that can be used in the invention is a p-glucanosyltransferase. Such an enzyme may be capable of catalyzing the degradation of (1,3) (1,4) glucan and / or cellulose and / or a cellulose degradation product.

A "glucuronidase" includes enzymes that catalyze the hydrolysis of a glucuronoside, for example p-glucuronoside, to produce an alcohol. Many glucuronidases have been characterized, and may be suitable for use in the invention. For example p-glucuronidase (EC 3.2.1.31), hyalurono-glucuronidase (EC 3.2.1.36), glucuronosyl disulfoglucosamine glucuronidase (3.2.1.56), glycyrrhizinate p-glucuronidase (3.2.1.128) or aD-glucuronidase (EC 3.2.1.139 ).

A composition for use in the invention may comprise an expansin or expansin-like protein, such as swollenin (see Salheimo et al., Eur. J. Biohem. 269, 4202-4211,2002) or a swollenin-like protein.

Expansins are involved in the loss of cell wall structure during plant cell growth. Expansins have been proposed to alter the hydrogen bond between cellulose and other cell wall polysaccharides without having hydrolytic activity. In this way, they are thought to allow the cellulose fibers to slip and the cell wall to enlarge. Swollenin, an expansin-like protein, contains an N-terminal 1 carbohydrate binding module (CBD) family domain and a C-terminal expansin-like domain. For the purposes of this invention, an expansin-like protein or swollenin-like protein can comprise one or both of such domains and / or can alter cell wall structure (such as altering cellulose structure), optionally without producing detectable amounts of reducing sugars.

A composition for use in the invention may include a cellulose-induced protein, for example the polypeptide product of the c ip l or cip2 gene or similar genes (see Foreman et al., J. Biol. Chem. 278 (34), 31988- 31997, 2003), a cellulose / cellulosome integrating protein, for example the polypeptide product of the cipA or cipC gene, or a scallop or a scallopkin-like protein. Scallops and cellulose integrating proteins are multifunctional integrating subunits that can organize cellulolytic subunits into a multi-enzyme complex. This is achieved by interaction of two complementary classes of domain, that is, a cohesion domain in scallopkin and a dockerin domain in each enzyme unit. The scallop subunit also carries a cellulose binding module (CBM) that mediates the binding of the cellulosome to its substrate. A scallop or cellulose integrating protein, for the purposes of this invention, may comprise one or both of such domains.

A composition for use in a method of the invention may be composed of one member of each of the aforementioned classes of enzymes, several members of one class of enzyme, or any combination of these classes of enzymes or helper proteins (i.e. the proteins mentioned in this document that do not have enzymatic activity per se, but that nevertheless help in lignocellulosic degradation), as long as they fall under the definitions of the claims.

A composition for use in a method of the invention, as defined in the claims, may be composed of enzymes from (1) commercial suppliers; (2) cloned genes that express enzymes; (3) complex broth (such as that resulting from the growth of a microbial strain in a medium, in which the strains secrete proteins and enzymes into the medium; (4) cell lysates from cultivated strains as in (3); and / or ( 5) Plant Material Expressing Enzymes Different enzymes in a composition of the invention can be obtained from different sources.

Enzymes can be produced either exogenously in microorganisms, yeasts, fungi, bacteria or plants, then they are isolated and added, for example, to a lignocellulosic raw material. Alternatively, enzymes are produced, but not isolated, and raw cell-mass fermentation broth, or plant material (such as corn stubble or wheat straw), and the like, may be added, for example, to the raw material. Alternatively, the crude cell mass or enzyme production medium or plant material can be treated to prevent further microbial growth (eg, by heating or adding antimicrobial agents), then can be added to, eg, a raw material . These crude enzyme mixtures can include the organism producing the enzyme. As an alternative, the enzyme can be produced in a fermentation using a raw material (pretreated) (such as corn stubble or wheat straw) to provide nutrition to an organism that produces an enzyme or enzymes. In this way, the plants that produce the enzymes can themselves serve as a lignocellulosic raw material, and can be added to the lignocellulosic raw material.

In the uses and methods described herein, the components of the compositions described above can be provided simultaneously (i.e. as a single composition per se) or separately or sequentially.

Lignocellulosic material

The lignocellulosic material herein includes lignocellulosic and / or hemicellulosic material. Lignocellulosic material suitable for use as a raw material in the invention includes biomass, for example virgin biomass and / or non-virgin biomass such as agricultural biomass, commercial organic matter, construction and demolition debris, urban solid debris, waste paper and garden debris. . Common forms of biomass include trees, shrubs, and lawns, wheat, wheat straw, sugarcane bagasse, rod grass, miscanthus, corn, corn stubble, corn husks, corn cobs, canola stalks, seed stalks soybeans, sweet sorghum, corn kernels, including grain fibers, grain mill products and by-products such as corn, wheat, and barley (including wet milling and dry milling) often referred to as "bran or fiber," as well such as urban solid remains, waste paper and garden remains. Biomass can also be, but is not limited to, herbaceous material, agricultural remains, forest remains, urban solid remains, waste paper, and remains of pulp and paper milling. "Agricultural biomass" includes branches, shrubs, reeds, corn and husks of corn, energy crops, woodlands, fruits, flowers, grains, grasses, herbaceous crops, leaves, bark, needles, logs, roots, young trees, timber crops of short rotation, bushes, stick grass, trees, vegetables, fruit peel, vines, sugar beet paste, wheat bran, oat husks, and hard and soft woods (not including woods with harmful materials). In addition, agricultural biomass includes organic waste materials generated from agricultural procedures including agricultural and forestry activities, specifically including forest wood debris. Agricultural biomass can be any of the above mentioned individually or any combination or mixture thereof.

Pretreatment

The raw material may optionally be pretreated with heat, mechanical and / or chemical modification, or any combination of such methods, to enhance the accessibility of the substrate to enzymatic hydrolysis and / or to hydrolyze hemicellulose and / or solubilize hemicellulose and / or cellulose and / or lignin, in any manner known in the art. In one embodiment, pretreatment is performed by treating lignocellulose with a steam explosion, hot water treatment, or dilute acid or dilute base treatment.

Washing stage

Optionally, the process according to the invention comprises a washing step. The optional washing step can be used to remove the water soluble compounds that can act as inhibitors for the fermentation step.

The washing step can be carried out in any known way.

Enzymatic hydrolysis

The enzyme composition used in the process of the invention can extremely efficiently hydrolyze lignocellulolytic material, for example corn stubble or wheat straw, which can then further be converted into a useful product, such as ethanol, biogas, butanol, acid lactic, a plastic, an organic acid, a solvent, a supplement for animal feed, a pharmaceutical, a vitamin, an amino acid, an enzyme or a chemical raw material. Additionally, intermediates from a post-hydrolysis process, for example lactic acid as an intermediate in biogas production, can be used as a building block for other materials. The present invention is exemplified by the production of ethanol, but this is done as an exemplification and not as a limitation of the other mentioned useful products that can be produced equally well.

The process according to the invention comprises an enzymatic hydrolysis step, as defined in the claims. Enzymatic hydrolysis includes, but is not limited to, hydrolysis for the purpose of liquefying the raw material, and hydrolysis for the purpose of releasing sugar from the raw material, or both. At this stage, the optionally pretreated and optionally washed lignocellulosic material is contacted with the enzyme composition according to the invention. Depending on the lignocellulosic material and the pretreatment, the different reaction conditions, for example temperature, enzyme dosage, hydrolysis reaction time, and dry matter concentration, can be tailored by experts to achieve the desired conversion of lignocellulose to sugar. Some indications are given below.

In one embodiment of the invention, hydrolysis is performed at a temperature of 45 ° C or higher, 50 ° C or higher, 55 ° C or higher, 602C or higher, 65 ° C or higher or 70 ° C or higher. High temperature during hydrolysis has many advantages, including working at the optimum temperature of the enzyme composition, reducing the risk of (bacterial) contamination, reduced viscosity, less required amount of cooling water, use of higher temperature cooling water, reuse of enzymes, and more.

In a further embodiment of the invention, the amount of enzyme composition added (also referred to herein as enzyme dosage or enzyme loading) is low. In one embodiment, the amount of enzyme is 6 mg protein / g dry matter by weight or less, 5 mg protein / g dry matter or less, 4 mg protein / g dry matter or less, 3 mg of protein / g of dry matter or less, 2 mg of protein / g of dry matter or less or 1 mg of protein / g of dry matter or less (expressed as protein in mg of protein / g of dry matter). In one embodiment, the amount of enzyme is 0.5 mg enzyme / g dry matter weight or less, 0.4 mg enzyme composition / g dry matter weight or less, 0.3 mg enzyme / g dry matter weight or less, 0.25 mg enzyme / g dry matter weight or less, 0.20 mg enzyme / g dry matter weight or less, 0.18 mg enzyme / g dry matter weight or less, 0.15 mg enzyme / g dry matter weight or less or 0.10 mg enzyme / g dry matter weight or less (expressed as the total cellulase enzymes in mg of enzyme / g of dry matter). Low enzyme dosage is possible, since due to the activity and stability of enzymes, it is possible to increase the hydrolysis reaction time.

In a further embodiment of the invention, the hydrolysis reaction time is 5 hours or more, 10 hours or more, 20 hours or more, 40 hours or more, 50 hours or more, 60 hours or more, 70 hours or more, 80 hours or more, 90 hours or more, 100 hours or more, 120 hours or more, 130 hours or more. In another aspect, the hydrolysis reaction time is 5 to 150 hours, 40 to 130 hours, 50 to 120 hours, 60 to 120 hours, 60 to 110 hours, 60 to 100 hours, 70 to 100 hours, 70 to 90 hours or 70 to 80 hours. Due to the stability of the enzyme composition, longer hydrolysis reaction times are possible, with correspondingly higher sugar yields.

The pH during hydrolysis can be chosen by an expert. In a further aspect of the invention, the pH during hydrolysis can be 3.0 to 6.4. The stable enzymes of the invention can have a wide pH range of up to 2 pH units, up to 3 pH units, up to 5 pH units. The optimal pH may be within the pH range 2.0 to 8.0, 3.0 to 8.0, 3.5 to 7.0, 3.5 to 6.0, 3.5 to 5.0 , 3.5 to 4.5, 4.0 to 4.5, or is approximately 4.2.

In a further embodiment of the invention, the hydrolysis step is carried out until 70% or more, 80% or more, 85% or more, 90% or more, 92% or more, 95 are released. % or more of the sugar available in the lignocellulosic material.

Significantly, a process of the invention can be performed using high levels of dry matter (of the lignocellulosic material) in the hydrolysis reaction. In this way, the invention can be carried out with a dry matter content of 10% by weight or more, about 11% by weight or more, about 12% by weight or more, about 13% by weight or more, about 14% in weight or greater, approximately 15% by weight or greater, approximately 20% by weight or greater, approximately 25% by weight or greater, approximately 30% by weight or greater, approximately 35% by weight or greater, or approximately 40% by weight or older. In a further embodiment, the dry matter content in the hydrolysis step is 14% by weight, 15% by weight, 16% by weight, 17% by weight, 18% by weight, 19% by weight, 20% by weight , 21% by weight, 22% by weight, 23% by weight, 24% by weight, 25% by weight, 26% by weight, 27% by weight, 28% by weight, 29% by weight, 30% by weight 31% by weight, 32% by weight, 33% by weight or more, or 14 to 33% by weight.

Fermentation

The process according to the invention can comprise a fermentation step. In one embodiment, the invention thus includes a process in which a microorganism is used for fermentation of a carbon source comprising a sugar or sugars, for example glucose, L-arabinose and / or xylose. The carbon source can include any oligo- or carbohydrate polymer comprising L-arabinose, xylose or glucose units, such as, for example, lignocellulose, xylans, cellulose, starch, arabinan and the like. For the release of the xylose or glucose units of such carbohydrates, appropriate carbohydrases (such as xylanases, glucanases, amylases, and the like) can be added to the fermentation medium, or can be produced by the modified host cell. In the latter case, the modified host cell can be genetically modified to produce and secrete such carbohydrases. An additional advantage of using oligo- or polymeric sources of glucose is that it makes it possible to maintain a low or lower concentration of free glucose during fermentation, for example using speed limiting amounts of carbohydrases. This, in turn, will prevent the suppression of the systems required for the metabolism and transport of sugars other than glucose such as xylose. In a preferred procedure, the modified host cell ferments both L-arabinose (optionally xylose) and glucose, preferably simultaneously, in which case a modified host cell that is insensitive to glucose repression is preferably used to prevent diauxic growth. In addition to a source of L-arabinose, optionally xylose (and glucose) as the carbon source, the fermentation medium will further comprise the appropriate ingredient required for the growth of the modified host cell. Compositions of the fermentation media for the growth of microorganisms such as yeast or filamentous fungi are well known in the art.

The fermentation time may be shorter than in conventional fermentation under the same conditions, in which part of the enzymatic hydrolysis still has to take place during fermentation. In one embodiment, the fermentation time is 100 hours or less, 90 hours or less, 80 hours or less, 70 hours or less, or 60 hours or less, for a sugar composition of 50 g / l glucose and other corresponding sugars of the lignocellulosic raw material (for example 50 g / l xylose, 35 g / l L-arabinose and 10 g / l galactose. more diluted sugar, the fermentation time can be correspondingly reduced.

The fermentation procedure can be an aerobic or anaerobic fermentation procedure. An anaerobic fermentation procedure is defined herein as a fermentation procedure performed in the absence of oxygen or in which oxygen is not substantially consumed, preferably less than 5, 2.5 or 1 mmol / l / h is consumed, more preferably 0 mmol / l / h (i.e. oxygen consumption is not detectable), and in which organic molecules serve both as electron donors and as electron acceptors. In the absence of oxygen, the NADH produced in glycolysis and biomass formation cannot be oxidized by oxidative phosphorylation. To solve this problem, many microorganisms use pyruvate or one of its derivatives as an electron and hydrogen acceptor, thus regenerating NAD +. Thus, in a preferred anaerobic fermentation process, pyruvate is used as an electron (and hydrogen) acceptor, and is reduced to fermentation products such as ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, butanol, p-lactam antibiotics and a cephalosporin. In a preferred embodiment, the fermentation procedure is anaerobic. An anaerobic procedure is advantageous since it is cheaper than aerobic procedures: less special equipment is needed. Additionally, anaerobic procedures are expected to give higher product performance than aerobic procedures. Under aerobic conditions, the biomass yield is normally higher than under anaerobic conditions. Consequently, normally under aerobic conditions, the expected product yield is less than under anaerobic conditions.

In another embodiment, the fermentation procedure is under limited oxygen conditions. More preferably, the fermentation process is aerobic and under limited oxygen conditions. An oxygen limited fermentation procedure is a procedure in which oxygen consumption is limited by the transfer of oxygen from the gas to the liquid. The degree of oxygen limitation is determined by the amount and composition of the incoming gas flow as well as the actual mixing / mass transfer properties of the fermentation equipment used. Preferably, in a process under limited oxygen conditions, the rate of oxygen consumption is at least 5.5, more preferably at least 6, and even more preferably at least 7 mmol / l / h.

The fermentation procedure is preferably performed at a temperature that is optimal for the modified cell. Thus, for most yeast or fungal cells, the fermentation procedure is performed at a temperature that is less than 42 ° C, preferably less than 382C. For yeast or filamentous fungal host cells, the fermentation procedure is preferably performed at a temperature that is less than 35, 33, 30, or 28 ° C, and at a temperature that is greater than 20, 22, or 25 ° C.

In one embodiment of the invention, fermentation is performed with a microorganism that is capable of fermenting at least one C5 sugar. In one embodiment, the process is a process for the production of ethanol, whereby the process comprises the step of fermenting a medium containing a sugar or sugars with a microorganism that is capable of fermenting at least one C5 sugar, thereby that the host cell is capable of fermenting glucose, L-arabinose and xylose to ethanol. In one embodiment thereof, the microorganism that is capable of fermenting at least one C5 sugar is a yeast. In one embodiment, the yeast belongs to the genus Saccharomyces, preferably of the species Saccharomyces cerevisiae, in which genetic modifications have been made. An example of such a microorganism and its preparation is described in more detail in WO 2008/041840. In one embodiment, the fermentation procedure for ethanol production is anaerobic. The term anaerobic has been previously defined here. In another preferred embodiment, the fermentation procedure for the production of ethanol is aerobic. In another preferred embodiment, the fermentation process for the production of ethanol is under limited oxygen conditions, more preferably aerobic and limited oxygen conditions. The limited oxygen conditions have already been defined here above.

In such a procedure, the volumetric productivity of ethanol is preferably at least 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0 or 10.0 g of ethanol per liter per hour. The yield of ethanol over L-arabinose and optionally xylose and / or glucose in the process is preferably at least 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95 or 98%. Ethanol yield is defined here as a percentage of the theoretical maximum yield, which, for glucose and L-arabinose and optionally xylose, is 0.51 g of ethanol per g of glucose or xylose.

In one aspect, the fermentation procedure leading to the production of ethanol has several advantages compared to known ethanol fermentation procedures:

- anaerobic procedures are possible;

- limited oxygen conditions are also possible;

- higher ethanol yields and ethanol production rates can be obtained;

- the strain used may be able to use L-arabinose and optionally xylose.

As an alternative to the fermentation procedures described above, at least two different cells can be used, this means that this procedure is a co-fermentation procedure. All preferred embodiments of the fermentation procedures as described above are also preferred embodiments of this co-fermentation procedure: identity of the fermentation product, identity of the source of L-arabinose and the source of xylose, fermentation conditions ( aerobic or anaerobic conditions, limited oxygen conditions, temperature at which the procedure is being performed, ethanol productivity, ethanol yield).

The fermentation procedure can be performed without any requirement to adjust the pH during the procedure. That is, the procedure is one that can be carried out without the addition of any acid or base. However, this excludes a pretreatment step, in which acid can be added. The point is that the composition for use in the invention is capable of acting at a low pH and, therefore, there is no need to adjust the acid pH of a raw material pretreated with acid so that saccharification or hydrolysis can take place . Accordingly, a method of the invention may be a zero waste method using only organic products that do not require an inorganic chemical input. It should be noted that the fermentation process is only part of the invention in combination with the present hydrolysis process, as defined in the claims.

Overall reaction time

In accordance with the invention, the overall reaction time (or reaction time of the hydrolysis step and the fermentation step together) can be reduced. In one embodiment, the overall reaction time is 300 hours or less, 200 hours or less, 150 hours or less, 140 hours or less, 130 or less, 120 hours or less, 110 hours or less, 100 hours or less, 90 hours or less, 80 hours or less, 75 hours or less, or approximately 72 hours at a glucose yield of 90%. Correspondingly shorter overall times can be achieved at a lower glucose yield.

Fermentation products

Fermentation products that can be produced according to the method of the invention include amino acids, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical raw materials, plastics, solvents, fuels or other organic polymers, lactic acid. and ethanol, including ethanol as a fuel (the term "ethanol" being understood to include ethyl alcohol or mixtures of ethyl alcohol and water).

Some specific value-added products that can be produced by the methods of the invention include, but are not limited to, biofuels (including biogas, ethanol, and butanol); lactic acid; 3-hydroxy-propionic acid; acrylic acid; acetic acid; 1,3-propane-diol; ethylene; glycerol; a plastic; a specialty chemical; an organic acid, including citric acid, succinic acid, and maleic acid; a solvent; a supplement for animal feed; a pharmaceutical product such as a p-lactam antibiotic or a cephalosporin; a vitamin; an amino acid, such as lysine, methionine, tryptophan, threonine, and aspartic acid; an enzyme, such as a protease, a cellulase, an amylase, a glucanase, a lactase, a lipase, a lyase, an oxidoreductase, a transferase, or a xylanase; a chemical raw material; or a supplement for animal feed.

Separation of the fermentation product

The process according to the invention optionally comprises the recovery of the fermentation product. A fermentation product can be separated from the fermentation broth in any known way. For each fermentation product, the expert will therefore be able to select an appropriate separation technique. For example, ethanol can be separated from a yeast fermentation broth by distillation, for example steam distillation / vacuum distillation, in a conventional manner.

Use of thermostable enzymes under optimal temperature conditions

In one embodiment, the method uses thermostable enzymes, such as Rasamsonia cellulolytic enzymes , for the production of reducing sugars from a lignocellulosic raw material pretreated in, but not limited to, the production of ethanol. Rasamsonia cellulolytic enzymes applied to the pretreated lignocellulosic feedstock showed maximum conversion rates at a temperature within the range of 50 to 70 ° C. The enzyme remains active in these circumstances for 14 days and more, without a complete cessation of activity.

Using optimal temperature conditions, a maximum amount of reducing sugars can be released from the raw material (total hydrolysis) within the shortest possible hydrolysis time. In this way, 100% conversion of cellulose to glucose is achieved in less than 5 days.

The theoretical maximum yield (Yps max in g of product per gram of glucose) of a fermentation product can be obtained from a biochemistry textbook. For ethanol, 1 mol of glucose (180 g) produces, according to the normal glycolysis fermentation route in yeast, 2 mol of ethanol (= 2x46 = 92 g of ethanol. The theoretical maximum yield of ethanol on glucose is per both 92/180 = 0.511 g of ethanol / g of glucose.

For butanol (PM 74 g / mol) or iso butanol, the theoretical maximum yield is 1 mol of butanol per mol of glucose. So Yps max for (iso-) butanol = 74/180 = 0.411 g of (iso-) butanol / g of glucose.

For lactic acid, the fermentation yield for homolactic fermentation is 2 moles of lactic acid (MW = 90 g / mol) per mol of glucose. According to this stoichiometry, the Yps max = 1 g of lactic acid / g of glucose.

For other fermentation products, a similar calculation can be made.

The cost reduction achieved with the application of Rasamsonia cellulolytic enzymes will be the result of an overall reduction in procedure time.

Compensating for lower enzyme dosage with prolonged hydrolysis time using Rasamsonia enzymes

Due to the high stability of stable enzymes, activities do not cease over time, although less reducing sugars are released in the course of hydrolysis. It is possible to reduce the dosage of enzyme and prolong the use of the enzyme by prolonging the hydrolysis times to obtain similar levels of reducing sugars released. For example, 0.175 ml of enzyme / g of raw material dry matter resulted in the release of approximately 90% of the theoretical maximum of reducing sugars from pretreated raw material in 72 h. When 0.075 ml of enzyme / g of raw material dry matter are used, approximately 90% of the theoretical maximum conversion is achieved in 120 h. The results show that, due to the stability of the enzyme activity, the reduction of the enzyme dosage can be compensated by prolonging the hydrolysis time to obtain the same amount of reducing sugars. The same is true for the hydrolysis of pretreated raw material at dry matter contents greater than 10%, which shows a prolonged hydrolysis time compensation effect at 15% of dry matter raw material.

The cost reduction achieved using stable cellulolytic enzymes, such as from Rasamsonia, results from requiring less enzyme dosing, resulting in similar hydrolysis conversion yields.

Risk reduction on contamination with stable enzymes

In a common procedure for converting lignocellulosic material to ethanol, the process steps are preferably performed under septic conditions to reduce operating costs. Therefore, contamination and growth of contaminating microorganisms may occur and result in undesirable side effects, such as production of lactic acid, formic acid and acetic acid, loss of yield of ethanol on the substrate, production of toxins and extracellular polysaccharides, which can significantly affect production costs. A high process temperature and / or a short process time will limit the risk of contamination during hydrolysis and fermentation. Thermostable enzymes, like those of Rasamsonia, are capable of hydrolyzing a lignocellulosic raw material at temperatures higher than 60 ° C. At these temperatures, the risk of a contaminating microorganism causing unwanted side effects will be very small to almost zero.

During the fermentation stage, in which ethanol is produced, temperatures are typically between 30 and 37 ° C, and preferably will not rise due to production losses. By applying fermentation procedure times as short as possible, the risks and effects of contamination and / or growth of contaminants will be reduced as much as possible. With stable enzymes, such as those from Rasamsonia, fermentation times as short as possible can be applied (see description above), and in this way the risks of contamination and / or growth of contaminants will be reduced as much as possible. In this way, the cost reduction achieved with the application of Rasamsonia thermoset cellulolytic enzymes will result in a lower risk of process failure due to contamination.

Stable enzymes reduce cooling costs and increase productivity of ethanol plants

The first stage after heat pretreatment will be to cool the pretreated feedstock to temperatures where enzymes are optimally active. On a large scale, this is typically done by adding (chilled) water, which, apart from lowering the temperature, will reduce the dry matter content. Using thermostable enzymes, such as those from Rasamsonia, a reduction in costs can be achieved by the fact that (i) less cooling of the pretreated raw material is required since higher temperatures are allowed during hydrolysis, and (ii) less water will be added , which will increase the dry matter content during hydrolysis and fermentation and, in this way, will increase the production capacity of ethanol (amount produced per unit time per volume) of an ethanol plant. Also, by using thermostable enzymes, such as those from Rasamsonia, cost reduction can also be achieved by using cooling water that has a higher temperature than water used in a process with a non-thermostable enzyme.

Recycling of enzymes after hydrolysis with stable enzymes

At the end of hydrolysis, the activities of the enzymes appear to be low since few reducing sugars are released once almost all the cellulose has been converted. However, the amount of enzyme activity present has decreased only a little, presumably mainly due to the absorption of enzymes in the substrate. By applying solid-liquid separation after hydrolysis, such as centrifugation, filtration, sedimentation-settling, etc., 60% or more can be recovered, for example 70% of the enzymatic activity in solution, and reused for the hydrolysis of a new lignocellulosic raw material pretreated during the next hydrolysis.

Furthermore, after solid-liquid separation, the enzyme in solution can be separated from the solution containing reducing sugars and other products of hydrolysis of the enzymatic actions. This separation can be carried out by means of, although without limitation (ultra and micro) filtration, centrifugation, sedimentation-settling, sedimentation, with or without first adsorption of the enzyme to a support of any kind.

For example, after hydrolysis of raw material pretreated with 0.175 ml / g enzyme load as raw material dry matter for 20 h, 50% of the theoretical maximum amount of reducing sugars is released, and after the same hydrolysis over 72 h, 90% of the theoretical maximum amount of reducing sugars is released. By centrifugation and ultrafiltration, 60-70% of the enzyme activity was recovered in the retentate, while the filtrate contained more than 80% of the reducing sugars released. By reusing the retentate, either as is or after further purification and / or concentration, the enzyme dosage during the next hydrolysis step can be reduced by 60 to 70%. The cost reduction achieved using stable cellulolytic enzymes, such as from Rasamsonia, thus results in a lower dosage of enzymes being required.

Recycling of enzymes after hydrolysis in combination with enzyme production and recycling of yeast cells with stable enzymes

The procedure that includes the recycling of enzymes after hydrolysis, as described above, can be combined with the recycling of the ethanol-producing microorganism after fermentation and with the use of the filtrate containing reducing sugars as substrate (purified and / or concentrated or diluted) in fermentation for enzyme production and as a substrate for the cultivation of ethanol-producing microorganisms.

Recycling of enzymes after vacuum distillation with stable enzymes

The thermostability of enzymes such as those of Rasamsonia, causes remaining cellulolytic activity after hydrolysis, fermentation and vacuum distillation in the fine processing residues. Total enzyme activity is reduced during the three successive process steps. Therefore, the fine process residues obtained after vacuum distillation can be reused as an enzyme source for a newly started hydrolysis-fermentation-distillation process cycle of converting pretreated wheat straw to ethanol. The fine processing residues can be used as a concentrated or (undiluted) and / or purified form and with or without additional enzyme supplements.

Recycling of enzyme in combination with enzyme supplementation after vacuum distillation with thermostable enzymes

In an optimal procedure, an amount of enzyme is supplemented in the fine processing residues, before being reused in a new procedure cycle, equal to the amount of activity lost during the three successive procedure steps of the previous procedure cycle. In this way, enzyme overdosing is avoided and therefore a more efficient use of the enzyme is obtained.

Furthermore, by providing a high dosage of enzyme in the first process cycle, and supplementing enzyme in an amount equal to that of the activity lost during the three successive process steps in the subsequent procedure cycles, higher possible rates of hydrolysis can be obtained in each procedure cycle, resulting in short hydrolysis times of less than 48 h in combination with more efficient use of enzymes.

Use of stable enzymes in mixed systems

By applying mixing during hydrolysis, enzymes more often come into contact with substrates, resulting in more efficient use of catalytic activity. This will result in lower enzyme dosages and thus lower costs, unless mixing has a negative effect on enzymes. Stable enzymes, such as Rasamsonia's thermostable enzymes , are robust and can withstand (locally) high shear and temperature circumstances, which is the case during intensive mixing of suspensions. The use of this in mixed systems is therefore beneficial and will lead to dosing and therefore cost reduction.

Examples

Experimental information

Strains

The Rasamsonia {Talaromyces) emersonii strain was deposited at CENTRAAL BUREAU VOOR SCHIMMELCULTURES, Uppsalalaan 8, PO Box 85167, NL-3508 a D Utrecht, The Netherlands in CBS Accession Number 393.64.

Other suitable strains can also be used in the present examples to show the effect and advantages of the invention. For example TEC-101, TEC-147, TEC-192, TEC-201 or TEC-210 are suitable Rasamsonia strains that are described in WO2011 / 000949.

Preparation of an acid pretreated corn stubble substrate

Diluted acid pretreated corn stubble (aCS) was obtained as described in Schell, D.J., Applied Biochemistry and Biotechnology (2003), vol. 105-108, p. 69-85. A pilot scale pretreatment reactor operating under steady state conditions of 190 ° C, residence time of 1 min., And an effective H2SO4 concentration of 1.45% (w / w) in the liquid phase was used .

Protein measurement assays

1. Total protein

TCA biuret

The method was a combination of protein precipitation using trichloroacetic acid (TCA) to remove nuisance substances and allow determination of protein concentration with the Biuret colorimetric reaction. In the Biuret reaction, a copper (II) ion is reduced to copper (I), which forms a complex with the nitrogens and carbons of the peptide bonds in an alkaline solution. A violet color indicates the presence of proteins. The intensity of the color and, therefore, the absorption at 546 nm is directly proportional to the protein concentration, according to the Beer-Lambert law. Normalization was performed using BSA (Bovine Serum Albumin), and the protein content was expressed in g of protein as equivalents of BSA / l or mg of protein as equivalents of BSA / ml. Protein content was calculated using standard calculation protocols known in the art, by plotting OD546 against the concentration of samples with known concentration, followed by calculating the concentration of unknown samples using the equation generated from the line calibration.

2. Individual proteins using PAGE

Sample pretreatment by SDS-PAGE

Based on the estimated protein concentration of the samples, the following sample preparation was performed. To 10 µl of sample, 40 µl of MilliQ water and 50 µl of TCA (20%) were added to dilute the sample five times (~ 1 mg / ml) and precipitate the proteins. After 1 hour on ice, the sample was centrifuged (10 minutes, 14000 rpm). The pellet was washed with 500 pl of acetone and centrifuged (10 minutes, 14000 rpm). The pellet was treated as described below.

SDS-PAGE

The pellet was dissolved in 65 µl of MilliQ water, 25 µl of NuPAGE ™ LDS sample buffer (4x) from Invitrogen and 10 µl of NuPAGE ™ sample reducing agent (10x) from Invitrogen. Before the denaturation step, the sample was diluted 5-fold using a MilliQ mix; NuPAGE ™ LDS Sample Buffer and 10 pl NuPAGE ™ Sample Reducer in 65:25:10 ratio. After mixing, the samples were incubated in a thermal mixer for 10 minutes at 70 ° C. Sample solutions were applied on a 4-12% Bis-Tris gel (NuPAGE ™ BisTris, Invitrogen). A sample (10 pl) of M12 marker (Invitrogen) was also applied on the gel. The gel was used at 200 V for 50 minutes, using XCELL Surelock, with 600 ml of 20 x diluted SDS buffer in the external buffer chamber and 200 ml of 20 x diluted SDS buffer, containing 0.5 ml of antioxidant (NuPAGE ™ Invitrogen) in the internal buffer chamber. After use, the gel was rinsed twice with demineralized water, and the gels were fixed with 50% methanol / 7% acetic acid solution for one hour and stained with Sypro Ruby (50 ml per gel) overnight. . An image was formed using Typhoon 9200 (610 BP 30, Green (532 nm), PMT 600V, 100 microns) after washing the gel with MilliQ water.

Quantitative protein analysis

Using the Typhoon scanner, the relationship between protein bands within a row was determined using standard methods known in the art. The sample was applied in triplicate, and the gray values were determined using the Image quant program. Values are expressed as relative% protein to total protein, calculated using the gray value of the selected protein band relative to the total gray value of all protein bands.

Glucan Conversion Calculation:

% conversion of glucan (%) = (glucose (g / l) x 100%) / (glucan (fraction in DM) x dm (g / kg) x 1.1) in which:

glucose (g / l) = concentration of glucose in the supernatant after hydrolysis.

glucan (fraction in dm) = glucan content of the substrate before pretreatment.

dm (g / kg) = dry matter of the hydrolysis (f.i. 20% dm = 200 g / kg).

1.1 = weight gain due to the incorporation of water during hydrolysis.

Calculation example:

glucose = 60 g / l

glucan fraction = 0.40 (it is 40% in dry matter)

dm = 200 g / kg

glucan conversion example = (60 * 100) / (0.4 x 200 x 1.1) = 68% conversion

Example 1

Evaluation of the effect of the absence of oxygen during hydrolysis on the cellulolytic activity of enzyme cellulase cocktails

The effect of oxygen absence during hydrolysis on the cellulolytic activity of three different enzyme cocktails was evaluated according to the procedures described below. Hydrolysis reactions were performed with an acid pretreated corn stubble raw material (aCS) at a final concentration of 10% w / w DM. This raw material solution was prepared by diluting a concentrated raw material solution with water. Subsequently, the pH was adjusted to a pH of 4.5 with a 4M NaOH solution. The removal of oxygen from the raw material was accomplished in two stages. First, the raw material solution was degassed by vacuum ultrasound in an ultrasound bath (Bransonic 5510E-DTH, fit; Degas) for 15 minutes. In the second stage, the oxygen was further removed by continuous spraying of a nitrogen stream through a 500 ml solution of the 10% DM raw material over a period of 3 hours. Before being sprayed through the raw material solution, the nitrogen stream was sprayed through water to saturate it with steam and prevent evaporation of the water from the raw material solution. In parallel, 500 ml of the same batch of 10% w / w DM aCS were sprayed with air as a control sample containing oxygen in a similar setup and according to the same protocol.

Hydrolysis of the oxygen-depleted (nitrogen-sprayed) and oxygen-saturated (air-sprayed) 10% w / w feedstock solutions were performed in air-tight 30 ml centrifuge bottles (Nalgene Oakridge) in a total reaction volume of 10 ml. The flasks, which already contained the cellulase solution, used for the oxygen-depleted experiment were sprayed with nitrogen before and during filling of the same with the raw material. Each hydrolysis was performed in duplicate with 7.5 mg / g cellulase enzyme cocktail DM added in a total volume of not more than 375 ml. The three cellulase enzyme cocktails tested included: a TEC-210 mix (cellulase mix), a 4E-GH61 mix (consisting of 9% w / w total BG protein, 30% w / w total CBHI protein, 25 % w / w total CBHII protein and 36% w / w total GH61 protein) and a 4E-EG mixture (consisting of 9% w / w total BG protein, 30% w / w total CBHI protein, 25 % w / w of total CBHII protein and 36% w / w of total protein EG). TEC-210 was fermented according to the inoculation and fermentation procedures described in WO2011 / 000949. Mix 4E was used (as described in WO2011 / 098577).

The centrifuge flasks containing the raw material and the enzyme solution were placed in an oven incubator (Techne HB-1D hybridization oven) and incubated for 72 hours at 65 ° C while being spun at set point 3 (12 rpm per minute). After hydrolysis, the samples were chilled on ice and immediately 50 ml of each supernatant was diluted in 1450 ml of grade I water. The subsequently diluted supernatant was filtered (0.45 mm filter, Pall PN 454), and the Filters were analyzed to determine the sugar content as described below.

The sugar concentrations of the diluted samples were measured using an HPLC kit equipped with an Aminex HPX-87P column (Biorad # 1250098) by elution with water at 85 ° C at a flow rate of 0.6 ml per minute, and quantified by integration of glucose signals from detection of refractive index (RI) calibrated with glucose standard solutions.

The data presented in Table 1 / Figure 1 show that the glucose released from the nitrogen sprayed raw materials is less than the glucose released from the air sprayed raw materials, both for incubations of the TEC-210 mix and the 4E mix. -GH61. There is no detectable difference in glucose release between the nitrogen and air sprayed raw materials for the samples hydrolyzed by the 4E-EG mix.

Based on these results, it is concluded that the presence of oxygen improves the cellulolytic behavior of cellulase mixtures that contain GH61 enzymes.

Table 1: The effect of spraying nitrogen or air through a 10% aCS raw material before hydrolysis, on the total amount of glucose released by three different cellulase mixtures

Example 2

The effect of oxygen on the cellulolytic activity of cellulase enzyme cocktails during the hydrolysis of lignocellulosic raw materials

This example shows the effect of oxygen on the cellulolytic activity of the enzyme cocktail during the hydrolysis of the lignocellulosic raw material. The hydrolysis reactions are carried out with an acid pretreated corn stubble raw material (aCS) at a final concentration of 20% w / w DM. This raw material solution is prepared by diluting a concentrated raw material solution with water. Subsequently, the pH is adjusted to pH 4.5 with a 10% (w / w) NH4OH solution.

Hydrolysis is carried out in a stirred reactor with controlled pH and controlled temperature, with a working volume of 1 l. Each hydrolysis is performed in duplicate with 2.5 mg / g of DM of TEC-210 cellulase enzyme cocktail. TEC-210 was produced according to the inoculation and fermentation procedures described in WO2011 / 000949.

The following experiments are carried out:

1. 1 l of 20% aCS, pH 4.5, temperature 62 ° C, stirrer speed 60 rpm (this corresponds to a DO level of <0.002 moles of oxygen per m3), 2.5 mg / g of dm of the TEC-210 cellulase cocktail, incubation time 120 hours (reference experiment).

2. Like Experiment 1, but at the start of hydrolysis, air spraying into the solution started at a dissolved oxygen level of 20% (this corresponds to 0.03 mol oxygen per m3, measured using a DO electrode (dissolved oxygen)). This level of dissolved oxygen is maintained throughout the remainder of the hydrolysis procedure.

3. As experiment 1, but after 72 hours air is sprayed into the solution starting at a dissolved oxygen level of 20% (this corresponds to 0.03 moles of oxygen per m3, measured using a DO (oxygen) electrode dissolved)). This level of dissolved oxygen is maintained throughout the remainder of the hydrolysis procedure.

After hydrolysis, the samples are chilled on ice and immediately 50 µl of each supernatant is diluted in 1450 µl of grade I water. The subsequently diluted supernatant is filtered (0.45 pm filter, Pall PN 454), and the Filters are analyzed to determine the sugar content as described below.

The sugar concentrations of the diluted samples are measured using an HPLC kit equipped with an Aminex HPX-87P column (Biorad # 1250098) by elution with water at 85 ° C at a flow rate of 0.6 ml per minute, and quantified by integration of glucose signals from detection of refractive index (RI) calibrated with glucose standard solutions.

The results, visible in Figure 2, clearly show increased glucose production in the case of air addition. Furthermore, the air added to the hydrolysis reaction in the second part of the time demonstrates a higher glucose production compared to not adding air or adding air during the entire hydrolysis step.

Example 3

The effect of partial aeration (over time) on the enzymatic hydrolysis of lignocellulosic raw material on a pilot scale

This example shows the effect of the dissolved oxygen concentration on the cellulolytic activity of the cocktail or enzyme composition during the hydrolysis of lignocellulosic raw material on a pilot scale. The reactions of Hydrolysis is performed with an acid pretreated corn stubble raw material (aCS) at a final concentration of 17.1% w / w DM. The raw material solution is prepared by diluting a concentrated raw material suspension with water. The pH is adjusted to pH 4.5 with a 25% (w / w) NH ⁴ OH solution.

Enzymatic hydrolysis is performed in a 270 liter pilot scale reactor with controlled pH and temperature, with a working volume of 150 liters. Oxygen dissolved during the procedure is controlled by adjusting the impeller speed to a given air flow and overpressure. The enzymatic hydrolysis is carried out at a dosage of 2.5 mg (TCA protein) / g of dm TEC-210 cellulase enzyme cocktail. TEC-210 was produced according to the inoculation and fermentation procedures described in WO011 / 000949.

The following experiments are carried out:

Experiment 1

Aeration from 0 to 120 hours: 150 l of 17.1% pCS, pH 4.5, temperature 62 ° C, 1 bar overpressure, air flow rate 10 kg / h in the headspace, 2.5 mg of TCA / gm dm TEC-210 cellulase cocktail, incubation time 120 hours in a 270 liter pilot scale reactor. The dissolved oxygen (DO) concentration of the reaction mixture was constantly measured using a DO electrode. The DO was controlled to a level of 0.15-0.22 moles / m ³ by adjusting the impeller speed.

Experiment 2

Aeration between 72 and 120 hours: 150 l of 17.1% pCS, pH 4.5, temperature 62 ° C, an enzyme dosage of 2.5 mg TCA / g of cellulase cocktail dm TEC-210 and a total incubation time of 120 hours in a 270 liter pilot scale reactor. The dissolved oxygen (DO) concentration of the reaction mixture was constantly measured using a DO electrode. During the first 72 hours of the procedure, the following settings were applied: no overpressure, no airflow in the headspace, and the DO is controlled to a level of [0.02-0.05] moles / m3 by adjusting the impeller speed. During the last 48 hours of the procedure, the following settings were applied: an overpressure of 1 bar, an air flow of 10 kg / h in the headspace, and the DO was controlled at a level of 0.15-0, 22 moles / m ³ by adjusting the impeller speed.

During enzymatic hydrolysis, samples were taken daily for analysis of carbohydrates (glucose, cellobiose) by NMR and measurement of viscosity and pH.

Composition analysis of pretreated corn stubble was performed by chemical hydrolysis of the sample and determination of monosaccharides by NMR.

Samples taken during enzymatic hydrolysis were analyzed for (oligo) sugars, organic acids and flow NMR inhibitors.

The results are presented in Figure 4, and show that, during enzymatic hydrolysis, in Experiment 2 with partial aeration (□ = aeration between hydrolysis time is 72 and 120 hours), more glucose is produced than during enzymatic hydrolysis in experiment 1 (■ = aeration between hydrolysis time is 0 and 120 hours).

Example 4

The effect of the timing of the dissolved oxygen supply on the enzymatic hydrolysis of the lignocellulosic raw material

This example shows the effect of the timing of the dissolved oxygen supply on the enzymatic hydrolysis of the lignocellulosic raw material. Hydrolysis reactions are carried out with an acid pretreated corn stubble (aCS) raw material at a final concentration of 20% w / w DM. The raw material solution is prepared by diluting the concentrated raw material suspension with water. The pH is adjusted to pH 4.5 with a 25% (w / w) NH ⁴ OH solution.

Enzymatic hydrolysis is performed in a 2-liter reactor that has controlled pH and temperature, with a working volume of 1 liter. Dissolved oxygen during the procedure is controlled by adjusting the impeller speed and continuous cooling of the headspace with fresh air in the case of increased dissolved oxygen concentration. The enzymatic hydrolysis is carried out at a dosage of 1.5 mg (TCA protein) / g of dm of TEC-210 cellulase enzyme cocktail. TEC-210 was produced according to the inoculation and fermentation procedures described in WO2011 / 000949.

The following experiments are carried out:

Experiment 1. Aeration from 0 to 7 hours: 1 l of 20% pCS, pH 4.5, temperature 62 ° C, 1.5 mg TCA / g of cellulase cocktail dm TEC-210, incubation time 120 hours. The dissolved oxygen (DO) concentration of the reaction mixture was constantly measured using a DO electrode. DO was controlled at a level of> 0.05 mol / m ³ during the first 7 hours of the hydrolysis procedure. Between 7 and 120 hours after the hydrolysis time, the DO was maintained at a level <0.02 mol / m ³ .

Experiment 2. Aeration between 72 and 120 hours: 1 l of 20% pCS, pH 4.5, temperature 622C, 1.5 mg TCA / g of cellulase cocktail dm TEC-210, incubation time 120 hours. The dissolved oxygen (DO) concentration of the reaction mixture was constantly measured using a DO electrode. DO was controlled at a level of <0.01 mol / m3 during the first 72 hours of the hydrolysis procedure. Between 72 and 120 hours of the hydrolysis time, the DO was maintained at a level> 0.05 mol / m3.

During enzymatic hydrolysis, samples were taken daily for analysis of carbohydrate (glucose, cellobiose) by NMR and measurement of viscosity and pH.

The composition analysis of the pretreated corn stubble was performed by chemical hydrolysis of the sample and determination of the monosaccharides by NMR.

The results are presented in Figure 5 and clearly demonstrate an increase in the rate of glucose formation when the reaction mixture is aerated. Experiment 1, which aired between 0 and 7 hours, clearly shows an increased glucose formation rate during the first 7 hours of the procedure compared to the non-aerated situation during that phase of the procedure of Experiment 2. In addition, Experiment 2 demonstrates an increase in the rate of glucose formation between 72 and 120 hours compared to the non-aerated situation during that period in Experiment 1.

Claims (13)

1. Procedure for the preparation of a sugar product from lignocellulosic material, comprising the following steps: a) optionally, pretreatment of the lignocellulosic material, b) optionally, washing the optionally pretreated lignocellulosic material, c) enzymatic hydrolysis of the optionally washed and / or optionally pretreated lignocellulosic material using an enzyme composition comprising at least two cellulases and whereby the enzyme composition comprises at least GH61, and d) optionally, recovery of the sugar product, wherein, for part of the enzymatic hydrolysis time, oxygen is added to the lignocellulosic material, and for part of the enzymatic hydrolysis time less or no oxygen is added to the lignocellulosic material compared to the other part of the hydrolysis time enzymatic, the reactor for enzymatic hydrolysis has a volume of 1 m3 or more, and the dry matter content in the hydrolysis step (c) is 10% by weight or more, in which the part of the time in which Adds more oxygen is 12 to 50% when oxygen is added in the second half of the enzymatic hydrolysis, or 2 to 30% of the total time of the enzymatic hydrolysis when oxygen is added in the first half of the hydrolysis time enzymatic, and so the concentration of oxygen in the liquid phase of hydrolysis during the part of the time in which oxygen is added is at least 2 times the concentration of oxygen in the liquid phase during the part of time in which it is added. add e less or no oxygen. 2. Procedure for the preparation of a fermentation product from lignocellulosic material, comprising the following steps: a) optionally, pretreatment of the lignocellulosic material, b) optionally, washing the optionally pretreated lignocellulosic material, c) enzymatic hydrolysis of the optionally washed and / or optionally pretreated lignocellulosic material using an enzyme composition comprising at least two cellulases and whereby the enzyme composition comprises at least GH61, d) fermentation of the hydrolyzed lignocellulosic material, to produce a fermentation product, and e) optionally, recovery of the fermentation product, wherein, for part of the enzymatic hydrolysis time, oxygen is added to the lignocellulosic material, and for part of the enzymatic hydrolysis time less or no oxygen is added to the lignocellulosic material compared to the other part of the hydrolysis time enzymatic, the reactor for enzymatic hydrolysis has a volume of 1 m3 or more, and the dry matter content in the hydrolysis step (c) is 10% by weight or more, in which the part of the time in which Adds more oxygen is 12 to 50% when oxygen is added in the second half of the enzymatic hydrolysis, or 2 to 30% of the total time of the enzymatic hydrolysis when oxygen is added in the first half of the hydrolysis time enzymatic, and so the concentration of oxygen in the liquid phase of hydrolysis during the part of the time in which oxygen is added is at least 2 times the concentration of oxygen in the liquid phase during the part of the time in which it is added. add e less or no oxygen 3. Process according to claim 1 or 2, in which the part of the time in which less or no oxygen is added is 10 to 80% of the time of the total enzymatic hydrolysis. 4. Process according to any of claims 1 to 3, in which the part of the time when oxygen is added, the concentration of oxygen in the liquid phase, in which the lignocellulosic material is present during enzymatic hydrolysis, is at least 0.001 moles / m3 or at most 0.12 moles / m3. 5. Process according to any of claims 1 to 4, wherein the oxygen is in the form of bubbles. 6. Process according to any of claims 1 to 5, wherein the enzymatic hydrolysis time is 5 to 150 hours. 7. Process according to any of claims 1 to 6, wherein the enzyme composition used retains activity for 30 hours or more. 8. Process according to any of claims 1 to 7, wherein the hydrolysis is carried out at a temperature of 45 ° C or more. 9. Process according to any one of claims 1 to 8, wherein the enzyme composition is derived from a fungus, or the enzyme composition comprises a fungal enzyme. 10. Process according to any of claims 1 to 9, wherein the dry matter content in the hydrolysis step (c) is 14% by weight or more. 11. A process according to any one of claims 1 to 10, wherein the enzymatic hydrolysis takes place in a batch, and / or continuous batch culture reactor. 12. A process according to any one of claims 1 to 11, wherein oxygen is introduced as an oxygen containing gas. 13. A process according to any of claims 2 to 12, wherein the fermentation is carried out with a microorganism that is capable of fermenting at least one C5 sugar.